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Gene Therapy with a Lentiviral Vector in Adrenoleukodystrophy

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Hematopoietic Stem Cell Gene Therapy with a Lentiviral Vector in X-Linked Adrenoleukodystrophy

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Lentiviral-mediated gene therapy of hematopoietic stem cells delays disease progression in patients with a fatal brain disorder.

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Department of Research and Development, uniQure Biopharma B.V., Amsterdam, Netherlands

Gene therapy is an emerging and powerful therapeutic tool to deliver functional genetic material to cells in order to correct a defective gene. During the past decades, several studies have demonstrated the potential of AAV-based gene therapies for the treatment of neurodegenerative diseases. While some clinical studies have failed to demonstrate therapeutic efficacy, the use of AAV as a delivery tool has demonstrated to be safe. Here, we discuss the past, current and future perspectives of gene therapies for neurodegenerative diseases. We also discuss the current advances on the newly emerging RNAi-based gene therapies which has been widely studied in preclinical model and recently also made it to the clinic.

AAV Gene Therapy

Gene therapy is an emerging therapeutic tool used to deliver functional genetic material to cells in order to correct a defective gene. By delivering a copy of a therapeutic gene to affected cells, the product encoded by that gene [i.e., its messenger RNA (mRNA) and/or proteins] will be continuously synthesized within the cell, utilizing the cell's own transcriptional and translational machinery ( Porada et al., 2013). The main advantage of this technology is that it offers a potentially life-long therapeutic effect without the need for repeated administration. Gene therapy can be used to correct defective genes by introducing a functional copy of the gene, by silencing a mutant allele using RNA interference (RNAi), by introducing a disease-modifying gene, or by using gene-editing technology ( Grimm and Kay, 2007; Dow et al., 2015; Saraiva et al., 2016).

Gene therapy vectors can be either viral or non-viral. Different physical and chemical systems can be applied to deliver therapeutic genes to cells without the need of a viral vector. Non-viral vectors have no size limitation for the therapeutic gene, generally have a low immunogenicity risk, and can be produced at relatively low costs ( Nayerossadat et al., 2012). However, due to the fact that high therapeutic doses are required when using non-viral technologies, and the resulting gene expression is generally transient, most gene therapies now rely on viral vectors. Numerous viral vector types have been tested in clinic, including vaccinia, measles, vesicular stomatitis virus (VSV), polio, reovirus, adenovirus, lentivirus, γ-retrovirus, herpes simplex virus (HSV) and adeno-associated virus (AAV) ( Lundstrom, 2018). Vaccinia, measles, VSV, polio, reovirus, adeno, and HSV vectors are currently mainly used in either vaccines or cancer therapeutics, while lentiviral and γ-retroviral vectors are predominantly used for transduction of transplantable cells ( Finer and Glorioso, 2017). For in vivo gene delivery, AAV is currently the preferred vector.

Adeno-associated virus belongs to the Parvoviridae family and is preferred for gene therapy because it is non-replicating (AAV requires a helper virus for replication), has a low immunogenicity profile, and is not known to cause disease ( Aschauer et al., 2013; Naso et al., 2017). In the absence of a helper virus, AAV may stably integrate into the host genome, but at a relatively low frequency. The genome of wild-type AAV is about 4.7 kb and is flanked between two inverted terminal repeats (ITRs) ( Figure 1A) ( Ojala et al., 2015). The open reading frame between the ITRs contains a replication (Rep) gene and a capsid (Cap) gene. The ITRs are cis-acting elements and are required for genome replication, integration, and packaging into the capsid. The Rep gene encodes four proteins (Rep78, Rep68, Rep52, and Rep40) that have important roles in replication and encapsidation of the viral DNA ( Collaco et al., 2003). The Cap gene encodes three capsid proteins (VP1, VP2, and VP3) and an assembly activating protein that promotes capsid formation ( Sonntag et al., 2010). There is a growing number of naturally occurring and engineered AAV serotypes with different viral capsids that have altered tissue tropism, transduction rate, or other features such as the ability to cross the blood-brain barrier ( Zincarelli et al., 2008; Deverman et al., 2016). Recombinant AAV (rAAV) can be produced by replacing the Rep and Cap genes with an expression cassette containing a therapeutic gene of interest ( Figure 1B). Formation of wild-type AAV is prevented by expressing the Rep and Cap genes on a separate plasmid (AAV packaging plasmid or AAV helper plasmid). The ITRs are the minimal regions required to be retained in rAAV to allow packaging of its genome. rAAV might still integrate randomly in the human genome at very low frequencies but most of its genome is maintained as episomal circular structures known as concatamers. rAAVs have been widely used in over 200 human clinical studies and they have shown to be safe ( Hudry and Vandenberghe, 2019). AAV-based gene therapies are highly attractive for the treatment of neurodegenerative diseases due to the neuronal tropism and their good safety profile demonstrated in clinical studies. In addition, a single administration results in long-term, potentially life-long, gene expression which is a main advantage when in vivo AAV administration requires invasive procedures.

Figure 1. (A) Schematic of wild-type AAV. Its genome consists of the viral Rep and Cap genes flanked by two inverted terminal repeats (ITRs). (B) Schematic of recombinant AAV (rAAV) generated by replacing the viral genes for a therapeutic gene. The therapeutic transgene expression cassette consists of a promoter of choice, a therapeutic gene and a polyadenylation signal (not shown) and is flanked by the AAV ITRs. The Rep and Cap genes are expressed from a different plasmid or viral vector. The rAAV is generated by co-transfecting cells with the transgene cassette flanked by AAV ITRs, the Rep and Cap genes of a specific AAV serotype, and an adenovirus helper plasmid.

Neurodegenerative Diseases

Neurodegenerative diseases are a heterogeneous group of multi-system disorders affecting the central nervous system, ultimately leading to neurodegeneration ( Gitler et al., 2017). Examples of the most common neurodegenerative diseases are amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), spinocerebellar ataxias (SCAs), Huntington's disease (HD), Alzheimer's disease (AD), and Parkinson's disease (PD) ( Bertram and Tanzi, 2005). The prevalence of these largely age-dependent disorders is increasing, partly due to the aging population, which in turn places a major economic burden on health care services. Some neurodegenerative diseases are caused by genetic mutations and/or cellular and circuit dysregulation. In some cases, different neurodegenerative diseases are linked to the same polymorphisms or mutations, thereby sharing similar pathological mechanisms. It seems that certain environmental or lifestyle factors combined with genetic factors increase the risk for certain neurodegenerative disease but aging is considered to be the most important risk factor for the sporadic cases ( Brown et al., 2005). Although each neurodegenerative disease has a different pathophysiology, they all lead to damage to the nervous system due to features such as cell death, impaired/failure of axonal regeneration, demyelination, and/or structural and functional neuronal deficits ( Hussain et al., 2018). These pathological features occur in different combinations and their causes can be either genetic or unknown ( Hussain et al., 2018). Common underlying causes leading to these conditions can be attributed to abnormal accumulation of proteins such as amyloid in AD, misfolded proteins (typical for PolyQ diseases), aggregation of proteins such as Tau (AD and traumatic brain injuries), synuclein (PD) or TDP-43 (ALS), RNA toxicity, or translational products from repeats expansion within genes ( Blokhuis et al., 2013; Hussain et al., 2018). Each of these features has unique mechanisms of toxicity which in large part are currently not well-understood. Some mechanisms in pathogenesis of neurodegenerative diseases share key characteristics with prions suggesting that progression of certain neurodegenerative diseases may share similar mechanisms involved in prion diseases ( Frost and Diamond, 2010). For example, misfolded protein aggregates (such as Tau, α-synuclein, amyloid-ß, huntingtin) can spread via cell to cell interaction and invade healthy tissues. The misfolded proteins can induce secondary misfolding of other unrelated aggregation-prone proteins, impairing the entire proteostatic network ( Sweeney et al., 2017). Ultimately, they lead to a decline or even the complete loss of sensory, motor, and cognitive functions. Symptoms commonly associated with neurodegenerative diseases include cognitive impairment, memory loss, apathy, anxiety, muscle weakness, paralyzes, and difficulties with speech or breathing.

A major clinical challenge is the early diagnosis of neurodegenerative diseases, and due to overlapping symptoms discrimination between the different diseases is difficult. Moreover, early symptoms are often dismissed or interpreted as normal consequences of aging. Apart from diagnostic delay, additional challenges for new therapeutic approaches reaching the stage of clinical development include the lack of druggable targets, the limited choice of delivery methods, and a lack of reliable biomarkers and clinical parameters that predict therapeutic efficacy or the rate of disease progression. To date, neurodegenerative diseases cannot be cured, and only palliative treatments are available. As these diseases are devastating for patients and their families, in turn also causing a vast burden on society, there is an enormous need to better understand their causes, pathology, and clinical progression, and to develop early detection methods and new therapeutic interventions.

In this review we will provide an up-to-date overview of the development and characterization of gene therapies for neurodegenerative diseases with focus on two common neurodegenerative diseases, ALS and SCA3, each with a different pathology. In addition, we will outline the clinical studies in neurodegenerative diseases using adeno-associated viral vector (AAV) and will address some of the major translational challenges in this emerging therapeutic field.

Amyotrophic Lateral Sclerosis

ALS (or Lou Gehrig's disease) is the most common adult onset motor neuron disease affecting the upper- and the lower motor neurons in the brain and spinal cord, but other neuroanatomical regions may also be affected ( Geser et al., 2010; Oskarsson et al., 2018). The upper motor neurons are found in the motor cortex of the brain, while the lower motor neurons are located along the brainstem, spinal cord and extend to the muscles ( Ravits and La Spada, 2009). Degeneration of the upper motor neurons causes symptoms such as spasticity and hyperreflexia while the loss of lower motor neurons results in progressive muscle weakness, cramps, fasciculations, muscle wasting, and paralysis ( Ravits and La Spada, 2009; Ferrari et al., 2011). The prevalence of ALS is currently estimated at 5 in 100.000 but the estimated lifetime risk to develop the disease is about 1:400-800 ( Bennion Callister and Pickering-Brown, 2014; Ingre et al., 2015). The discordance between the low prevalence but high lifetime risk is explained by the fact that ALS patients have a very limited life span with a median survival ranging from 2 to 5 years from symptom onset ( Oskarsson et al., 2018). The understanding of the genetic causes of ALS is continually expanding, but our knowledge of other risk factors such as environmental factors, lifestyle, or aging remains poor. Only 10% of ALS cases are familial, and the causal genetic mutations are usually inherited in a mendelian autosomal dominant manner ( Siddique and Ajroud-Driss, 2011). Thus, most ALS cases are assumed to occur sporadically, and the main causes are still unknown.

Two drugs have been approved by the Food and Drug Administration for the treatment of ALS, but the efficacy of both drugs is modest. Riluzole - first approved in 1995 - is a glutamate receptor antagonist which may increase survival by 2 to 3 months. More than two decades later in 2017, Edaravone, a free radical scavenger was approved but its efficacy is still unclear, and at best the drug has a moderate effect on disease progression ( Cruz, 2018). More than 30 genes have been linked to ALS and mutations in chromosome 9 open reading frame 72 ( C9orf72), superoxide dismutase1 (SOD1), transactive response DNA-binding protein 43 (TDP-43), or fused in sarcoma (FUS) are responsible for most of the familial ALS cases. A hexanucleotide expansion consisting of GGGGCC (G ₄C ₂) nucleotides in the first intron of the C9orf72 gene is the most frequent genetic cause of ALS and is found in about 40-50% of familial ALS cases and 5-10% of sporadic ALS cases ( Umoh et al., 2016). The G ₄C ₂ repeat is transcribed bidirectionally and affected patients usually carry more than 30 G ₄C ₂ copies. Interestingly, the same mutation also causes FTD, the second most common form of dementia after AD. C9orf72 is responsible for 25% of familial FTD cases and 5-7% of sporadic FTD cases. ALS and FTD are considered overlapping diseases as about 15% of ALS patients develop FTD and up to 50% of ALS patients show some degree of functional loss in the frontal lobe of the brain ( Bennion Callister and Pickering-Brown, 2014). The function of the C9orf72 encoded protein is poorly understood, but it may be a regulator of the autophagy-lysosome pathway during nutrient stress responses ( Liu and Wang, 2019). There are at least three proposed pathogenic mechanisms in C9orf72 related ALS and/or FTD patients; (1) RNA-mediated toxicity, (2) repeat-associated non-ATG (RAN) translation, (3) haploinsufficiency. It is also possible that a combination of the three mechanisms contributes to the disease pathogenesis ( Figure 2) ( Kumar et al., 2017; Shi et al., 2018).

Figure 2. Mechanisms of toxicity associated with C9orf72 G ₄C ₂ repeat. (A) RNA-mediated toxicity. Repeat-containing sense and antisense RNA transcripts accumulate and sequester RNA binding proteins (B) RAN translation. The sense and antisense repeat-containing transcripts undergo RAN translation into five, potentially toxic DPRs (C) Haploinsufficiency. Hypermethylation of the expansion leads to reduced transcription of C9orf72.

RNA-Mediated Toxicity

RNA-mediated toxicity was first described in myotonic dystrophy type 1 (DM1), which is caused by a CTG repeat expansion in the 3′ UTR of the myotonic dystrophy protein kinase gene ( Mahadevan et al., 1992; Taneja et al., 1995). It was shown that RNA consisting of CUG repeats folds into stable structures that colocalize with RNA-binding proteins and sequester their function. For example, an important protein that is sequestered by CUG-containing RNA foci is the splicing factor muscleblind-like 1 (MBNL1) ( Mankodi et al., 2000). The sequestration of MBNL1 leads to its inactivation which subsequently causes mis-splicing of several pre-mRNAs, such as muscle-specific chloride ion channels and insulin receptors ( Mankodi et al., 2002; Dansithong et al., 2005). RNA foci are observed in several other neurodegenerative diseases caused by repeat expansions including ALS and SCA3 ( Belzil et al., 2013). In C9orf72-related ALS and FTD, accumulation of sense and antisense G ₄C ₂-containing RNA foci is detected in several brain-, spinal cord tissues, lymphocytes and fibroblasts. RNA foci are also detected in patient-derived induced-pluripotent stem cell (iPSC)-neurons. Although RNA foci mainly accumulate in the nucleus of cells they are also observed at lower concentrations in the cytoplasm. Several RNA binding proteins interact with G ₄C ₂ RNA repeats, such as ADARB2, hnRNPA1, hnRNPA1B2, Pur-α, FUS, Nucleolin, and TDP-43 ( Donnelly et al., 2013; Sareen et al., 2013; Kumar et al., 2017). However, the contribution of these proteins to neurodegeneration is only partially understood.

RAN Translation

Another proposed mechanism of toxicity in C9orf72 related ALS and/or FTD is through RAN translation ( Stepto et al., 2014; Peters et al., 2015; Kumar et al., 2017). It has been shown that the repeat-containing transcripts can be translated into dipeptide repeat proteins (DPRs) even in the absence of an ATG start codon and even though the mutation is in a non-coding region of C9orf72. These DPR proteins are toxic and they can form aggregates that accumulate in the brain and spinal cord of patients ( Zu et al., 2011; Zu et al., 2013). Six DPR proteins can be produced via unconventional translation in all reading frames. Glycine-alanine (GA) and glycine-arginine (GR) are produced from the sense repeat-containing transcripts, while proline-arginine (PR) and proline-alanine (PA) are produced from the antisense repeat-containing transcripts. Glycine-proline (GP) is produced from both sense and antisense repeat-containing transcripts ( Gitler and Tsuiji, 2016). Ample evidence indicates that DPR proteins are toxic and cause neurodegeneration. For example, neurotoxicity, proteasome activity, and endoplasmic reticulum stress were observed in primary neurons expressing GA proteins ( Zhang et al., 2014). The addition of recombinant GR and PR proteins to Hela cells and human astrocytes was toxic and caused alterations in RNA processing ( Gitler and Tsuiji, 2016). Expressing GR and PR proteins in Drosophila caused toxicity and early lethality ( Mizielinska et al., 2014). Several recent publications have demonstrated that DPR proteins can lead to the impairment of nuclear transport, causing accumulation of several RNA-binding proteins including TDP-43 in the cytoplasm ( Freibaum et al., 2015; Jovičič et al., 2015; Zhang K. et al., 2015). Cytoplasmic TDP-43 aggregation is observed in ∼97% of cases of ALS, including those associated with C9orf72 mutations. TDP-43 protein is predominantly found in the nucleus but can shuttle between the nucleus and cytoplasm to regulate processes such as RNA processing, transcription, pre-mRNA splicing, transport and stabilization of mRNA ( Chen-Plotkin et al., 2010; Warraich et al., 2010; Moujalled et al., 2017). In patients, TDP-43 accumulates as cytosolic inclusions with a C-terminal fragment of 25 or 35 kDa. The aggregated TDP-43 is also post-translationally modified, being heavily ubiquitinated and phosphorylated at the C-terminal region ( Moujalled et al., 2017). Notably, mutations in TARDBP, the gene that encodes the TDP-43 protein, have also been linked to ALS, but it remains unclear whether it is depletion of nuclear TDP-43, gain of a toxic function of cytoplasmic TDP-43, or a combination of these processes that are neurotoxic.

Furthermore, it has been recently demonstrated that expressing PR in mice also resulted in neurodegeneration and premature death ( Zhang et al., 2019). PR proteins localized to the heterochromatin caused abnormal histone H3 methylation and aberrations in nuclear lamins and heterochromatin protein 1α (HP1α). This resulted in down-regulation of numerous differentially expressed genes and upregulation of many repetitive elements accompanied by the accumulation of double-stranded RNA, ultimately leading to neuronal death ( Zhang et al., 2019). Thus, as RNA foci and DPRs both contribute to the pathogenesis of ALS, their inhibition could potentially reduce the disease burden in patients.

Haploinsufficiency

The thought that haploinsufficiency may contribute to C9orf72-related ALS originates from the finding that both lower RNA and protein levels have been reported in brain and spinal cord tissues and in iPSC-neurons derived from ALS patients. Several mechanisms may lead to reduced levels of C9orf72, including abortive transcription caused by G-quadruplexes and R-loop structures of the repeat-containing transcripts. In addition, the C9orf72 locus is hypermethylated in several mutation carriers, which can lead to epigenetic silencing. Similar mechanisms of gene silencing have been observed in other repeat expansion diseases such as Friedrich ataxia and fragile X mental retardation syndrome. However, haploinsufficiency alone does not explain the observed ALS pathology in patients. Several loss-of-function mutations in C9orf72 have been identified and appeared to be non-pathogenic ( Harms et al., 2013). Furthermore, complete C9orf72 knockout mice showed no neurodegeneration but rather splenomegaly, enlarged cervical lymph nodes, and autoimmune related premature death ( Koppers et al., 2015; Jiang et al., 2016). Interestingly, this phenotype was rescued in mice hemizygous for C9orf72, suggesting that a partial knockdown of the gene would be well-tolerated ( Jiang et al., 2016). A recent publication suggests a cooperative pathogenesis between gain- and loss-of function mechanisms ( Shi et al., 2018). Reduced C9orf72 protein in cultured motor neurons caused accumulation of glutamate receptors and excitotoxicity in response to glutamate. In addition, these motor neurons showed impaired clearance of DPRs and were hypersensitive for these proteins.

As most evidence points toward a toxic gain of function resulting from this mutation, current therapeutic strategies are mainly based on silencing of the mutated gene. For example, with antisense oligonucleotides (ASOs) or RNAi ( Lagier-Tourenne et al., 2013; Martier et al., 2019a, b).

Spinocerebellar Ataxia Type 3

The ATXN3 gene encodes a 42-kDa protein called ataxin-3 which consists of an N-terminal catalytic Josephin domain and two to three (dependent on the splice variant) C-terminal ubiquitin (Ub)-interacting motifs flanking the polyQ tract ( Paulson et al., 1997, 2017; Evers et al., 2014). The ataxin-3 protein is widely expressed in different cell types of peripheral and neuronal tissues and present in both nucleus and cytoplasm ( Macedo-Ribeiro et al., 2009). Ataxin-3 is believed to interact with up to 100 proteins involved in ubiquitin-dependent pathways and quality control. The protein has a role in various ubiquitin-dependent pathways that maintain protein homeostasis. By partnering with ubiquitin ligases, ataxin-3 may be able to regulate, or edit, the lengths and linkage types of ubiquitin chains on proteins, and in this manner either rescue proteins from being degraded or stimulate protein breakdown ( Burnett et al., 2003; Todi et al., 2009). Additional roles in endoplasmic reticulum-associated degradation, aggresome (aggregates of misfolded proteins) production and DNA repair have also been implicated ( Wang et al., 2012; Chatterjee et al., 2015; Gao et al., 2015; Ashkenazi et al., 2017; Minoia et al., 2017).

Mechanisms of Toxicity

Figure 3. Ataxin-3 mediated mechanisms of toxicity. (A) RNA toxicity. RNA containing the CUG repeats can sequester function of transcription factor (B) Mutant ataxin-3 toxicity. The CAG repeat-containing ATXN3 transcript is translated into a protein with a polyQ expansion. Proteolytic cleavage of mutant ataxin-3 can generate C-terminal protein fragments containing the polyQ repeat. The mutant ataxin-3 and C-terminal protein fragments can both cause several cellular disturbances such as transcriptional deregulation, impaired autophagy, mitochondrial dysfunction, proteasomal impairment, compromised axonal transport, and DNA damage.

In addition to the mechanisms described above, which are based on protein toxicity, RNA toxicity could also contribute to the disease. As mentioned before, RNA containing CUG repeats can sequester various transcription factors and undergo RAN translation. A crucial piece of evidence for RNA toxicity was observed in nematodes. CAG repeats cloned into the 3′ UTR of a marker protein showed severe toxicity in a length-dependent manner in Caenorhabditis elegans (C. elegans). The CUG RNAs formed RNA foci and colocalized with C. elegans muscleblind protein. The highest CAG repeats were embryonically lethal while the shorter CAG repeats were tolerated ( Wang et al., 2011; Nalavade et al., 2013). Since the mutated ATXN3 gene produces a protein with a toxic gain of function, it is considered a promising target for gene silencing approaches such as ASOs and RNAi-based gene therapy ( Evers et al., 2014; Toonen et al., 2017; Martier et al., 2019c).

In vivo Models for ALS and SCA3

Model systems that recapitulate the different aspects of neurodegenerative diseases are essential to understand the pathology, and to predict clinical efficacy during early development of new therapeutic approaches. Development of animal models for neurodegenerative diseases is a major challenge as the diseases have complex pathologies and symptoms can take decades to unfold. As a result, most of the currently available animal models for neurodegenerative diseases are not exact phenotypes of the human diseases and therefore lack the ability to forecast clinical success of new therapeutic approaches. Moreover, studies performed in vitro and in vivo on cells and small animals are difficult to translate to larger animals, because factors such as delivery route, dosing, distribution, and toxicity vary between the different model systems ( Joshi et al., 2017).

Animal Models for C9orf72 ALS/FTD

Various mouse models have been generated for C9orf72 ALS/FTD that recapitulate distinct disease-related pathological, functional, and behavioral phenotypes. An overview of the currently available models is shown in Table 1. The first mouse model for C9orf72 ALS was created in 2015 by somatic transduction of the C57BL/6J mouse CNS using an AAV carrying 66 G ₄C ₂ repeat ( Chew et al., 2015). Although this model did not develop the severe ALS/FTD symptoms, some important features were observed that correlate with the pathology in patients. For example, sense RNA foci and DPRs produced from RAN translation were observed and TDP-43 inclusions were found in ∼7-8% of neurons in the cortex and hippocampus ( Chew et al., 2015). In addition, mild neurodegeneration but no motor neuron loss was observed ( Chew et al., 2015). Four other transgenic mouse models were created expressing either full length or truncated human C9orf72 with the G ₄C ₂ expansion. The C9orf72 Exon 1-6 BAC (G ₄C ₂)500 SJL/B6 mice expressed sense and antisense RNA foci and poly GP proteins but no motor and/or behavioral impairment was observed ( Peters et al., 2015). The Tg( C9orf72_3) line 112 mouse was created containing the full length human C9orf72 with multiple G ₄C ₂-repeat sizes ranging from 100 to 1000 repeats ( O'Rourke et al., 2015). This model showed sense and antisense RNA foci and mild accumulation of poly GP but despite some evidence for nuclear stress there was no neuronal loss observed. Similarly, the BAC-C9-450 model also produced sense and antisense RNA foci in some brain regions and in the spinal cord as well as DPR proteins with an age dependent increase of poly(GA). Despite a partial learning deficit and increased anxiety, no other motor or behavioral changes were observed. In contrast, the FVB/NJ-Tg( C9orf72)500Lpwr/J mouse model expressing the full length mutated human C9orf72 gene including the 52 kb 5′ upstream region and the 19 kb 3′ downstream region of the gene did show progressive neurodegeneration and decreased survival which is also seen in ALS patients ( Liu et al., 2016). Sense RNA foci were observed in almost all NeuN positive neurons in the cortex, hippocampus and cerebellum. Antisense RNA foci were predominantly found in the motor cortex, hippocampus, cerebellar Purkinje layer and interneurons in the lateral and posterior horn of the spinal cord. DPR proteins in this mouse model increased with age throughout the brain, and nuclear and cytoplasmic TDP-43 aggregates were mainly observed in degenerating brain regions. The severe phenotype was exclusively observed in about one third of the females, while most of the males and females developed a mild phenotype. Nevertheless, the observations in this model strongly support the involvement of the G4C2 repeat-mediated gain of toxicity in the disease pathology.

Table 1. Overview of the currently available mouse models for C9orf72 related ALS and/or FTD.

Despite the variable penetrance, these models could still be valuable tools for research as they all display some specific features such as RNA foci and DPR proteins that are key characteristics of C9orf72 related ALS/FTD patients.

Animal Models for SCA3

More than 10 transgenic models for SCA3 have been published, all with variable differences in pathology and phenotype and most of them showing a mild form of neurodegeneration ( Gould, 2012). A summary of the most commonly used mouse models and their key characteristics is shown in Table 2. Three models showed a severe phenotype and will be discussed here in more detail. The first transgenic mouse model for SCA3 was created more than two decades ago expressing either full length or truncated ataxin-3 with 79 CAG repeat (Q79). A severe ataxic phenotype was observed only in the truncated model, with degeneration of the cerebellum and loss of Purkinje cells ( Ikeda et al., 1996). Due to this observation, it was strongly suggested that the C-terminal fragment of ataxin-3 containing the expanded polyQ could be more toxic by itself than when expressed as part of full length ataxin-3. Consistently, putative cleavage fragments of expanded ataxin-3 were identified in cell models, in post-mortem brain tissues of a SCA3 mouse model, in a drosophila model, and in patients ( Yamamoto et al., 2001; Berke et al., 2004; Goti, 2004; Colomer Gould et al., 2006; Jung et al., 2009). It is now well-accepted that the gain of toxicity is directly caused by the CAG repeat and that the affected neurons express a protease that cleaves the mutant ataxin-3 protein, releasing short soluble polyQ fragments that are highly neurotoxic and more prone to form aggregates ( Wang et al., 2011; Nalavade et al., 2013). Besides proteolytic cleavage of the C-terminal of the mutant ataxin-3 protein, mis-splicing could also play a role. A crucial evidence of mis-splicing leading to production of toxic short polyQ proteins was observed in HD. It was shown that exon 1 of the huntingtin, gene which contains the CAG repeat does not always splice to exon 2 but generates small polyadenylated HTTexon1 mRNA that encodes a small, highly pathogenic exon 1-polyQ HTT protein (also known as "exon-1 protein") ( Neueder et al., 2017).

Table 2. Overview of the currently available mouse models for SCA3.

The two other SCA3 mouse models (Q71C and 70.61) with a severe phenotype both express mutant ataxin-3 under control of the prion protein promoter. Within 1 to 8 months, both models showed early onset and a rapidly progressive type of SCA3 with several abnormalities such as progressive neurodegeneration, weight loss, behavioral problems, neuronal inclusions and premature death. Taken together, all these models could be useful to further study the disease mechanism in SCA3 and serve as tools for preclinical studies targeting mutant ataxin-3. It should be noted that the models with a severe phenotype all use non-native promoters to drive expression of mutant ataxin-3, lack regulatory flanking sequences, and often contain an excessive number of transgene copies. Thus, these models do not exactly mimic the expression pattern of mutant ataxin-3 in patients.

RNAi-Based Therapeutic Strategies for Neurodegenerative Diseases

As discussed, several neurodegenerative diseases are caused by mutations that lead to gain of toxic functions, and gene silencing technologies are attractive to lower the expression of disease-causing genes. A specific technology that can be applied for gene silencing isRNAi.

The discovery of RNAi dates back almost three decades, when Napoli (1990) reported that injection of an extra chimeric copy of chalcone synthase, a gene responsible for the purple pigment anthocyanin in petunias, unexpectedly resulted in white petunias. Thus, instead of complementing each other and producing extra purple flowers, the two copies of chalcone synthase seemed to interact with each other and turn themselves off. The mechanism underlying this observation remained unclear until, when Fire et al. (1998) provided an explanation of the RNAi mechanism. They discovered that a small non-coding RNA, miRNA (lin-4), binds to the 3′-UTR of the lin-14 mRNA in C. elegans, silencing its expression. For this work they were awarded the Nobel prize in Physiology or Medicine in 2006 ( Fire et al., 1998). It was subsequently demonstrated that miRNA-mediated silencing also occurs in mammalian cells and this mechanism became a popular new tool to study gene function ( Elbashir et al., 2001).

It is now known that RNAi is a naturally occurring process in eukaryotic cells where double stranded RNA molecules can knock down or suppress the expression of genes that contain a homologous RNAi target sequence. RNAi plays an important physiological role in gene regulation and also has a function in the innate immune response of cells by providing protection against foreign nucleic acids from pathogens such as viruses and bacteria ( Ozcan et al., 2016). One of the first discovered mediators of RNAi is the RNAi-induced silencing complex (RISC), which has a nuclease activity that can cleave mRNA and knock down its expression. RNAi can be triggered by both endogenous and exogeneous double stranded RNAs ( Baulcombe, 1996; Montgomery and Fire, 1998; Saito et al., 2015). Three types of small non-coding RNAs use the RNAi pathway; microRNA (miRNAs), small interfering RNA (siRNA), and piwi-interacting RNAs (piRNAs). miRNAs and siRNAs have a role as negative regulators of gene expression, while the piRNAs defend organisms against transposable elements ( Aravin et al., 2007). miRNAs and siRNA are currently both being widely used as therapeutics in clinical trials and each system has its own merits.

miRNAs and Their Processing

miRNAs are small non-coding RNAs that are thought to regulate about 30% of genes in humans. More than 2000 miRNAs have been identified in humans and these are known to regulate several important cellular processes. Dysregulation of miRNAs is associated with several types of metabolic and CNS diseases, and with cancer. Apart from regulating gene expression, miRNAs can act as signaling molecules for intercellular communication. For example, it has been shown that miRNAs can be packaged into exosomes or microvesicles that, following secretion, can be endocytosed by secondary cells, and re-establish their function ( Kreth et al., 2018).

The biogenesis of a miRNA is tightly regulated in humans and involves 4 key enzymes: Drosha, exportin 5, Dicer, and argonaute (AGO) proteins ( Figure 4). The precursor of a miRNA is naturally encoded in the genome and is transcribed by RNA polymerase II or III into a long primary miRNA (pri-miRNA) with a cap and poly-A tail. The pri-miRNA transcript folds into a complex hairpin structure consisting of a double-stranded stem of about 30 bp, a terminal loop and two flanking unstructured single-stranded tails. The pri-miRNA is further processed in the cell nucleus by a ribonuclease called Drosha, resulting in a short 70-nt stem-loop structured precursor miRNA (pre-miRNA). The pre-miRNA is then transported to the cytoplasm by exportin 5 and is cleaved into a short double stranded miRNA by an RNAse III enzyme called Dicer. The double stranded miRNA is recognized by AGO proteins and is loaded on RISC, usually preserving the guide strand and degrading the passenger strand. The RISC-AGO complex guides the miRNA guide strand to its target mRNA and induces its degradation and/or inhibits its translation. The first seven nucleotides near the 5′ end of the guide strand form the seed sequence and are critical for the target recognition. It is also worth mentioning that not all miRNAs are processed as described above and miRNA processing in plants differs at several points from the above described pathway. Furthermore, several mammalian miRNAs can bypass Drosha and currently one miRNA is known to bypass Dicer processing ( Yang and Lai, 2011). This Dicer-independent miRNA is microRNA-451a (miR-451a or miR-451) and has a role in the regulation of erythroid development ( Herrera-Carrillo and Berkhout, 2017). Its recognition by Dicer seems to be perturbed due to its unusually short base paired stem and as a result, an active strand derived from the stem is produced, consisting of the single-stranded loop and part of the complementary stem region ( Figure 4). The ability to design artificial miRNAs that specifically silence disease-related genes is the basis for therapeutics such as miRNA mimics, anti-miRs, and artificial miRNAs. miRNA mimics are double stranded miRNAs made synthetically to match a corresponding miRNA aiming to compensate the loss of miRNAs that are downregulated in diseases. Anti-miRs are artificially made single stranded miRNA used to bind target miRNAs and inhibit their function. Artificial miRNAs are made by replacing the guide strand sequence of an endogenous miRNA precursor with the sequence of an mRNA of interest, enabling silencing of genes that are upregulated in diseases ( Figure 5). More recently, miR-451 was discovered that follows a non-canonical processing of miR-451 to silence genes involved in diseases ( Cheloufi et al., 2010; Cifuentes et al., 2010; Yang et al., 2010; Herrera-Carrillo and Berkhout, 2017). miR-451 produces no passenger strand as the pre-miR-451 escapes Dicer cleavage and only a 5′ arm strand is active on the targets. Thus, there is no miRNA duplex formation that requires strand separation and selection for the RISC. Because of this efficient processing, off-target effects due to a passenger strand activity can be neglected.

Figure 4. Schematic of miRNA processing pathway. Most miRNAs are processed in the Dicer dependent (canonical) pathway shown in the left part of the figure. After being transcribed, the pri-miRNA transcripts are cropped by Drosha in the nucleus, resulting in a 60-70 nt pre-miRNA. The pre-miRNA is exported to the cytoplasm by Exportin 5 and the hairpin is then diced by Dicer into ∼22-nt miRNA duplex, after which it is separated into a guide and passenger strand. The guide strand is usually loaded into Ago proteins to form RISC.

Figure 5. Artificial miRNA design. Artificial miRNAs can be made by replacing the mature miRNA sequence of a natural pri-miRNA for a complementary sequence of a target mRNA of interest. The artificial pri-miRNA sequence can be cloned in an expression construct. Upon transfection with the artificial miRNA construct, the artificial miRNA is processed in the cell into a mature artificial miRNA which can bind and knockdown expression of an mRNA of interest.

There are four Ago proteins and all are capable to repress translation or promote mRNA degradation. Ago2 is the only Ago protein with a slicer activity which plays a critical role in Dicer independent processing of miR451 (right part of the figure). miR451 is also processed in the nucleus by Drosha, but results in an unusually short 41-42-nt pre-miRNA which is not recognized by Dicer and does not form a miRNA duplex. However, the further processing of miR-451 requires the slicer activity of Ago2. Ago2 cleaves the 3′ arm of pre-miRNA by its slicer activity and yields a 30-nt intermediate whose 3′ end is further trimmed by PARN to generate a mature ∼23-nt miRNA. This mature miRNA is loaded into Ago proteins to form the RISC.

Clinical Applications for Therapeutic RNAi

Furthermore, Alnylam Pharmaceuticals was recently granted marketing authorization after a successful phase III clinical trial for GIVLAARI ® (givorisan), an aminolevulinate synthase 1 (ALAS1)-directed small interfering siRNA for treatment of Acute Hepatic Porphyria. Givorisan is injected subcutaneously once a month and its mode of action is to reduce elevated levels of ALAS1 mRNA and prevent accumulation of neurotoxic δ-aminolevulinic acid and porphobilinogen levels that are associated with acute porphyria attacks. The clinical trial demonstrated a 74% reduction of porphyria attack in patients, despite some frequently occurring adverse reactions such as injection site reaction, nausea, and fatigue ( Alnylam Pharmaceuticals, 2020).

Regulus therapeutics also have several RNAi-based therapeutics in the pipeline and currently RG-012 is in Phase II clinical trial. RG-012 is a single stranded, chemically modified oligonucleotide that binds to and inhibits the function of miR-21 for the treatment of Alport syndrome. Patients with Alport syndrome experience progressive loss of kidney function due to defective mutations in collagen genes which are normally responsible for maintaining the integrity and structure of the kidneys. MiR-21 represses expression of proteins in the PPARα signaling pathway which have a protective function in the kidneys. In preclinical studies, RG-012 showed strong inhibition of miR-21 and resulted in improvement of kidney function and an increased life expectancy ( Chau et al., 2012).

Another phase I/II clinical trial to treat patients with Hepatitis C infection based on shRNAs was conducted by Benitec Biopharma. Their lead product was TT-034 which is based on intravenous AAV8 delivery of three independent shRNAs targeting three well-conserved sequences located in the 5′ UTR and NS5B regions of the HCV genome ( Patel et al., 2016). Overall, TT-034 was well-tolerated and safe in patients. Furthermore, liver biopsies revealed sustained transduction of hepatocytes and expression of the three anti-HCV shRNAs. However, Benitec Biopharma decided to discontinue this program due to decrease in commercial opportunities for TT-034 following the introduction of highly effective viral eradication strategies based on (combinations of) small molecules.

Compared to miRNAs, siRNAs have been more widely tested in clinical trials for treatment of different types of diseases such as cancer, viral infections, age-related macular degeneration, diabetic macular, hypercholesterolemia, and ocular hypertension ( Chakraborty et al., 2017). DNA constructs encoding therapeutic RNAi following delivery by lentiviral vectors have also been clinically tested. Benitec, Inc. in collaboration with the City of Hope National Medical Center conducted a clinical trial to treat HIV-1 infection in AIDS/lymphoma patients ( DiGiusto et al., 2010). This was executed by genetically modifying hematopoietic stem cells (hSC) ex vivo by transduction with a lentiviral vector expressing three small RNAs targeting HIV: an shRNA targeting an exon in HIV-1 tat/rev, a RNA decoy for the HIV TAT-reactive element, and a ribozyme targeting the host cell CCR5 chemokine receptor. The transduced hSC were then infused into patients whose bone marrow had been ablated to treat their AIDS related lymphoma. Sustained expression of the anti-tat/rev shRNA and ribozyme was observed for up to 24 months post-infusion ( Tiemann and Rossi, 2009). Although this study demonstrated feasibility and safety of this approach, it failed to demonstrate clinical benefit because an insufficient number of hSCs were transduced.

In the neurodegenerative field, uniQure obtained Food and Drug Administration (FDA) clearance for AMT-130 which is the first AAV-miRNA-based gene therapy for a neurodegenerative disease to start a Phase I/II clinical trial. Recently, uniQure announced that the first two patients have been treated in this double-blind, randomized clinical trial being conducted in the United States. AMT-130 is based on a miRNA designed to target both wild-type and mutant Huntingtin allele (AAV5-miHTT) ( Miniarikova et al., 2016, 2017, 2018; Evers et al., 2018; Keskin et al., 2019). Significant lowering of human mutant huntingtin mRNA and protein was achieved in the brain of a transgenic HD (tgHD) minipig model after a single administration into the brain ( Evers et al., 2018). The Phase I/II clinical trial of AMT-130 for the treatment of HD will explore the safety, tolerability, and efficacy signals in 26 patients with early manifest Huntington's disease randomized to treatment with AMT-130 or an imitation (sham) surgery. The 5-year, multi-center trial consists of a blinded 18-month core study period followed by unblinded long-term follow-up. Patients will receive a single administration of AMT-130 through MRI-guided, convection-enhanced stereotactic neurosurgical delivery directly into the striatum (caudate and putamen). Additional details are available on www.clinicaltrails.gov (NCT04120493). A similar approach is being pursued by Voyager Therapeutics and promising results have been obtained with their lead candidate VY-HTT01 in preclinical models. miRNA mediated silencing is thought to be an attractive therapeutic modality in many other neurodegenerative diseases, including the SCAs, ALS, FTD, synucleopathies including Parkinson's disease, tauopathies and AD ( Xu et al., 2005; Do Carmo Costa et al., 2013; Liu et al., 2013; Nóbrega et al., 2013b). miRNAs may cause toxicity related to (passenger strand) off-target toxicity. Hence, during development these effects should be closely investigated and minimized, by selecting scaffolds with little to no passenger strand activity and by predicting the chances of binding to off-target genes using computer-based bioinformatic tools. Another important limitation of miRNA-based therapeutics is the required efficacy in the nucleus of cells due to the fact that active mature miRNAs resulting from canonical Dicer processing in the cytoplasm are thought not to re-enter the nucleus. However, as many of these hurdles are being tackled, more miRNA therapeutics are expected to reach the clinic.

Common Alternative Silencing Strategies for Neurodegenerative Diseases

Antisense Oligonucleotides

Antisense oligonucleotides (ASOs) are short, synthetic, single-stranded nucleic acids of about 20-25 bases long that bind cellular RNA and reduce, restore, or modify protein expression via several distinct mechanisms. The silencing pathway of ASOs differs from siRNA and miRNAs and there is no known cellular mechanism to facilitate strand recognition. ASOs classically bind to complementary mRNA through Watson Crick base-pairing leading to endonuclease-mediated transcriptional knockdown ( Rinaldi and Wood, 2018). Once bound to the mRNA ASOs can form an RNA-DNA hybrid that becomes a substrate for the enzyme RNase H, which hydrolyzes the mRNA resulting in its degradation ( Wu et al., 2004). The cleaved mRNA products are then processed by the normal cellular degradation pathways in the nucleus and cytoplasm ( Lima et al., 2016). ASOs can be engineered to have enhanced pharmacological properties by introducing backbone modifications. For example, modified ASOs can knockdown gene expression by sterically blocking splicing factors and altering pre-mRNA splicing, or by preventing ribosome recruitment to block mRNA translation ( Havens and Hastings, 2016). Interestingly, ASOs can destabilize splicing sites and displace or recruit splicing factors when designed to bind within intron-exon junctions. This makes it possible to exclude ("skip") or include exons of interest, which can be beneficial in genetic diseases ( Havens and Hastings, 2016). Through this mechanism normal gene function can be restored by re-establishing a normal reading frame following a pathogenic frame shift, or by excluding mutated segments of DNA ( Rinaldi and Wood, 2018). ASOs have been used in preclinical studies since the 1970s and several programs have made it into clinical development. A phase I ALS trial using intrathecal administration of ASOs targeting SOD1 was completed in 2012 by Ionis Pharmaceuticals ( Table 3) ( Miller T. et al., 2013; Miller T.M. et al., 2013). Unfortunately, this study failed to show reduction of SOD1 protein which was explained by the low target tissue ASO concentration. However, the treatment proved to be safe, and a follow up phase I/II trial study is ongoing (BIIB067; IONIS-SOD1). Ionis Pharmaceuticals also initiated a phase I clinical trial in 2019 with ASOs targeting C9orf72-ALS patients (BIIB078). BIIB078 is targeting specifically the intronic region of C9orf72 to cause reduction of only the repeat-containing C9orf72 transcripts and is administered intrathecally to adult patients. Ionis Pharmaceuticals has developed ASOs for many other diseases and have ongoing clinical trials for HD, myotonic dystrophy type 1 (DM1) and spinal muscular atrophy (SMA). Furthermore, several preclinical studies have shown efficacy of ASOs in C9orf72-related ALS and SCA3 in vitro and in vivo but these programs have not yet reached clinical development. A clinical study (NCT03508947) with ASOs that has raised some concerns regarding toxicity was performed by WAVE life Sciences to treat patients with Duchenne muscular dystrophy (DMD). DMD is a fatal genetic disorder characterized by progressive muscle wasting and is caused by mutations in the DMD gene which encodes for dystrophin, an essential protein to maintain muscle integrity. The ASO was administered intravenously and was designed to skip the mutated exon 51 in the DMD gene to restore normal function of dystophin ( Echevarría et al., 2018). Adverse events such as headache, fever, vomiting, and tachycardia occurred in the low and mid dose groups and these adverse events were even more severe in the high-dosed groups. WAVE life sciences reported plans to continue with the lower doses for Phase II/III trial but it is unclear if these adverse events will re-occur during re-administration and whether or not the lower doses are therapeutically effective. A major limitation of ASOs for clinical application is that they are degraded by endo- and exo-nucleases and re-administration is required ( Evers et al., 2015). This is especially problematic in CNS disorders that require invasive delivery methods. However, the pharmacological profiles of ASOs can be enhanced by introducing chemical modifications and efforts are currently being made to improve ASOs delivery to for example cross the blood-brain barrier (BBB) and to improve target engagement ( Juliano, 2016).

Table 3. Clinical trials using ASOs for ALS by Ionis Pharmaceuticals.

Gene Editing (CRISPR)

Gene editing is a relatively novel approach to remove, add, or alter DNA in a sequence-specific manner. The pivotal discovery that made gene editing possible was the discovery that the endogenous cellular repair machinery can be triggered by targeted DNA double strand breaks (DSBs) through homology-directed repair (HDR) or non-homologous end-joining (NHEJ). DNA DSBs can be introduced at a precise location by using engineered nucleases harboring a sequence-specific DNA-binding domain, fused to a DNA cleavage module. The most popular nucleases are zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the RNA-guide clustered regulatory interspaced short palindromic repeats (CRISPR) and CRISPR-associated system 9 (Cas9) (CRISPR/Cas9). ZFNs are currently the only gene editing technology that have made it to clinical trials. In 2018, Sangamo Therapeutics started a clinical trial using this technology in mucopolysaccharidosis II (MPS II). ZFNs consist of ∼30 amino acids which folds into ββα "fingers" structures that recognize and bind a trinucleotide sequence of DNA ( Gupta and Musunuru, 2014; Corrigan-Curay et al., 2015; Nature America, Inc, 2018). The nuclease domain is based on a restriction endonuclease ( FokI) that can cut DNA following dimerization ( Gupta and Musunuru, 2014). Binding to DNA sequences longer than 3 and up to 18 bp (in multiples of 3 bp) is possible by arranging a series of linked zinc-fingers. However, this requires recoding of proteins for each new target site which is a very challenging and time-consuming process. Another limitation is the restricted target site selection, as zinc-fingers can only target binding sites every ∼50-200 bp in a random DNA sequence ( Gupta and Musunuru, 2014). Furthermore, all gene editing technologies including zinc-fingers may have off-target effects, resulting in unwanted induction of DNA mutations or deletions. Like ZFNs, TALENs are based on a DNA-protein association but they use a different DNA binding domain termed (transcription activator-like effectors) TALE repeats ( Gupta and Musunuru, 2014). A TALE motif can recognize a single nucleotide and an array of TALEs can recognize and bind to a longer DNA sequence. Unlike ZFNs, the design of TALENs are less complicated and the TALE repeat array can be easily extended to whatever length is desired ( Gupta and Musunuru, 2014). Furthermore, as compared to ZFNs, site-specific targeting is easier because multiple TALEN pairs are available for each base pair of a random DNA sequence. Some major limitations for TALENs are off-target effects, and compared to ZFNs they are about 3x larger in size (∼3 kb) which makes delivery by viral vectors more challenging.

CRISPR/Cas9 is the latest of the above-mentioned nuclease systems and is based on a naturally occurring process in the adaptive immune system of the bacteria Streptococcus pyogenes. CRISPR/Cas9 is distinct from ZFN and TALEN endonucleases in that it does not use a protein but an RNA-guided system to perform genome editing. Unlike the other nuclease systems, CRISPR/Cas9 does not require custom design of novel proteins for each DNA target site. As a result, the design of constructs is relatively easy and cost-effective. Bacteria use the RNA-guided Cas proteins to create DSBs in exogenous DNA derived from invading viruses (bacteriophages). It does so by creating CRISPR arrays from exogenous DNA by insertion of these DNA sequences between short palindromic repeats in the CRISPR locus of its own DNA. Upon a repeated viral attack, these "foreign" inserts can be transcribed from the virus specific CRISPR locus into CRISPR RNA fragments that match the invading viral DNA. CRISPR RNAs contain a tail transcribed from the CRISPR locus which facilitates incorporation into complexes to form hairpin-like structures that allow them to dock on a Cas protein. The Cas protein is then guided by the CRISPR RNA to hybridize to its parental exogeneous DNA and induce a DSB ( Cong and Zhang, 2014). This CRISPR/Cas9 system can be implemented in eukaryotes by designing sequences that target a specific DNA sequence and co-expressing Cas9. The Cas9 protein cuts the DNA at the target site, which is repaired by HDR or NHEJ. Several animal models have been created using the CRISPR/Cas9 system and a rapidly increasing number of preclinical studies shows the promise of this technology in combination with gene therapy for the treatment of neurodegenerative diseases. For example, in AD, the defective (amyloid precursor protein) APP gene was successfully edited in human fibroblasts using a CRISPR/cas9 construct and this resulted in a reduction of the levels of Amyloid beta, a main component of plaques found in the brains of AD patients ( Gyorgy et al., 2016). In ALS, the G ₄C ₂ repeat in the non-coding region of the C9orf72 gene was successfully deleted in transfected patient-derived iPS cells and this prevented RNA foci formation as well as the promoter hypermethylation that is typical for ALS ( Pribadi et al., 2016). For HD, permanent suppression of mutant Huntingtin and it's aggregates was achieved in the striatum of the HD140Q-knock-in mice by CRISPR/Cas9 ( Yang et al., 2017). For SCA3, CRISPR/Cas9-mediated deletion CAG repeat in ATXN3 gene was successfully performed in patient-derived iPSCs ( Ouyang et al., 2018). Following the CAG deletion, the iPSCs retained pluripotency and neurons differentiated from these cells retained a normal ubiquitin-binding capacity of ATXN3.

The CRISPR/Cas9 system has great potential for targeting pathogenic genes, but major hurdles are the occurrence of off-target effects, Cas9 specificity, and potential mutagenesis. Not unexpectedly, continuous expression of Cas9 proteins at high concentrations has been linked to toxicity ( Morgens et al., 2017). The chances for off-target binding in the host genome using current systems are estimated to be relatively high (≥50%) ( Zhang X.H. et al., 2015). Another limitation is the delivery method as the CRISPR/Cas9 components are about 8-10 kb long whereas AAV vectors have a packaging capacity limited to 4.8 kb. Thus, the CRISPR/Cas9 system has a huge potential to be developed in therapeutic approaches but major challenges need to be overcome to make this technology suitable to treat human diseases.

AAVs for Gene Transfer to the Nervous System

A major difficulty to treat neurodegenerative diseases remains the method of administration. Due to existence of the BBB, many small molecules and most therapeutic nucleotides or gene therapy vectors that are administered systemically or orally fail to reach the brain or spinal cord at a therapeutically relevant dose. Different alternative routes of administration are being investigated for delivery to the CNS and their application is highly dependent on the disease pathology. While some diseases require local targeting or transduction, others require widespread distribution throughout the CNS. Intracerebral administration, directly injected in the parenchyma of the brain can be safely and effectively done using convection-enhanced delivery (CED) in combination with precise positioning using magnetic resonance imaging-based guidance technologies (MRI) ( Salegio et al., 2012). CED uses a pressure gradient to generate bulk flow within the brain parenchyma. Due to the low velocity of injection potential structural damage is minimized and a uniform distribution can be obtained ( Bankiewicz et al., 2000; Richardson et al., 2011). The advantage of this injection route is that it bypasses the BBB and high local transduction can be obtained with a relatively low dose of AAV and limited leakage to periphery organs. Further spread of AAV may occur via anterograde or retrograde transport along axons but this is highly dependent on the AAV serotype ( Aschauer et al., 2013; Warren Olanow et al., 2015). A drawback of this delivery route is that the procedure itself is invasive and may lead to complications such as bleeding or leakage of the administered vector into the CSF. In addition, intracerebral administration is only suitable for diseases with pathology limited to specific brain areas such as HD. In the current AMT-130 clinical trial by uniQure, the vector is delivered directly into the striatum (caudate and putamen) using MRI-guided, convection-enhanced stereotactic neurosurgery. This approach has been shown to be very precise and safe for direct infusion into the brain ( Samaranch et al., 2017). Alternative routes used to cover larger areas of the CNS are systemic or intrathecal delivery. Systemic delivery by intravenous injection is a relatively simple procedure, less invasive and avoids costly neurosurgical procedures. However, systemic delivery of AAV gene therapy is currently less suitable for neurodegenerative disorders because the bulk of such vectors is taken up by peripheral organs and can cause systemic immunogenicity. Even though some vectors can cross the BBB, the target tissue concentration after systemic delivery is often not therapeutically relevant. Nevertheless, a successful clinical trial with systemic administration was performed by AveXis to treat children with SMA ( Mendell et al., 2017). The vector used was a self-complementary AAV9 and following systemic administration a therapeutic effect in motor neurons in the spinal cord was observed. It remains questionable whether similar effects can be obtained in adults as the integrity of the BBB is known to be different. In addition to systemic administration, AveXis is currently performing another clinical trial in children with SMA by injecting intrathecally. In non-human primates (NHP), they showed improved transduction of the CNS with up to 10 times lower intrathecal dose as compared to intravenous administration ( Meyer et al., 2015). Intrathecal administration can be done through lumbar puncture, direct administration into the cisterna magna or in the cerebral ventricle ( Hocquemiller et al., 2016; Hinderer et al., 2017). Thus, the vector is delivered directly into the CNS and bypasses the BBB. This approach is less invasive than intracerebral administration and usually shows less leakage to the periphery organs compared to systemic administration. One limitation of intrathecal administration is dilution of vector and the consequent transduction is usually lower when compared to intracerebral administration. Furthermore, transduction of the deeper brain structures is poor, possibly because the vector needs to pass the ependymal cell layer or the pia mater ( Dindot et al., 2011; Mittermeyer et al., 2012).

Gene Therapy Clinical Trials for Neurodegenerative Diseases or Neuromuscular Diseases

Several gene therapies for different types of neurodegenerative diseases have progressed into clinical development. For C9orf72 related ALS and SCA3, no gene therapy has been clinically investigated yet. An overview of the currently ongoing clinical trials published on https://clinicaltrials.gov is depicted in Table 4 and will be further discussed in this paragraph.

Table 4. Gene therapy clinical trials for neurodegenerative/neuromuscular diseases.

Parkinson's Disease

Parkinson's disease (PD) is neurodegenerative disorder caused by loss of dopaminergic neurons in the substantia nigra, leading to bradykinesia, rigidity, tremor, and gait dysfunction. Expressing neurotrophic factors such as glial cell-derived neurotrophic factor (GDNF) were tested in a single clinical trial and neurturin (NTN) was investigated in three clinical trials in PD patients. The rationale for delivering these neurotrophic factors was not to target a causative pathological molecular pathway but to provide neurotrophic support to the degenerating neuronal population. Although AAV delivery of these neurotrophic factors were well-tolerated in patients, the efficacies are unclear. However, these studies were important to demonstrate the feasibility and tolerability of intraparenchymal delivery of gene therapy directly in the human brain.

Most gene therapy approaches for PD aim to restore dopamine production in neurons. Delivery of 1-amino acid decarboxylase (AADC), a key enzyme for dopamine production, showed to be more efficacious when compared to neurotrophic factors. Proof of concept studies in NHP based on delivery of AADC by AAV showed an increase in levels of dopamine which was sustained for up 7 years ( Bankiewicz et al., 2000, 2006). The first AAV delivered AADC study in humans (NCT00229736) was generally well-tolerated with some minor side effects ( Eberling et al., 2008; Christine et al., 2009). A significant improvement of Parkinson's Disease Rating Scale (UPDRS) score was reported at 6 months post-surgery and was sustained for up to 2 years. These promising results have triggered other trials based on AADC delivery and currently there are several clinical trials being conducted with higher doses and larger cohorts of patients. A clinical trial using a intrastriatal delivered lentivirus (ProSavin) expressing three key dopamine biosynthetic enzymes (tyrosine hydrolase, AADC, and GTP cyclohydrolase-1) for continuous dopamine production also demonstrated to be safe and well-tolerated with no surgical complications (CT01856439) ( Palfi et al., 2014; Piguet et al., 2017). A significant improvement of motor function was observed, and a long-term analysis is ongoing. Another lentiviral gene therapy that is currently being tested in clinic and is very similar to ProSavin also expresses tyrosine hydroxylase, AADC AND GTP-cyclohydrolase (NCT03720418). While there is no data available yet from this study, the preclinical results in human primary neuronal cultures and NHP showed higher dopamine production compared to ProSavin ( Badin et al., 2014).

Alzheimer's Disease

Alzheimer's disease (AD) is the most common cause of age-related dementia affecting more than 40% of individuals of 85 years and older. More than 100 therapeutic compounds have been tested to date, but all failed to positively modify the course of the disease. Most gene therapy clinical trials for AD are based on intracerebral delivery of AAV-NGF. NGF encodes for nerve growth factor and is similar to GDNF and NTN in that it could provide neurotrophic support to neurons. Preclinical studies have shown that NGF can prevent degeneration of adult cholinergic neurons in the fore brain after injury ( Kromer, 1987; Hefti, 2018). Although the surgical procedures and the treatments proved to be safe in humans, the efficacy of the studies were inconclusive. A more recent study for AD is based on intracisternal delivery of AAVrh.10hAPOE2 (NCT03634007). It was shown that inherence of an extra allele of APOE4 gene, a variant of apolipoprotein E (APOE), significantly increases the risk for developing sporadic AD. However, APOE2 homozygotes are protected against the disease and it is hypothesized that APOE2 delivery could be beneficial in APOE4 homozygous patients ( Rosenberg et al., 2018). Whether this approach indeed modifies the course of the disease is yet to be demonstrated.

Batten Disease

Batten disease or neuronal ceroid lipofuscinosis (NCL) is a rare group of neurodegenerative disorders that can manifest in infants, children, and adults. Symptoms include epilepsy, loss of cognitive and motor function, degeneration of the retina leading to blindness, and early death. More than a dozen CLN genes and more than 430 loss of function mutations have been linked to the disorders ( Mole and Cotman, 2015). AAV-based clinical trials for Batten disease have mainly focused on delivery of AAV-CLN2 and AAV-CLN3. The first study (NCT00151216), using intracranial delivery of AAV2-CUhCLN2 showed little therapeutic benefit. A follow up study is still ongoing and used a different vector (AAVRh.10CUhCLN2) but abnormal T2 hyperintensities was observed in first dosed patients. Therefore, the dose was lowered for the following patients. For CLN3, one study (NCT03770572) is still recruiting and is based on AAV9-CLN3 delivery. For CLN6, a study with intrathecally delivered scAAV9.CB.CLN6 is ongoing. According to a press release, preliminary data from the first two dosed patients showed no further progression of the disease (according to the Hamburg motor and language score) during the first 2 years after dosing.

Mucopolysaccharidoses (MPSs)

Mucopolysaccharidoses (MPSs) are rare metabolic diseases that are caused by impaired degradation of mucopolysaccharides, leading to accumulation of glycosaminoglycans in the lysosome. Symptoms can be detected as early as during the prenatal period but can also occur in adulthood. The symptoms in adults include difficulty of speech, ataxia, weakness, and dyskinesia, while children usually show neurological impairment, developmental delay, and premature death ( Platt, 2018). Several gene therapy clinical trials are currently in progress for the treatment of MPS I (Hurler infantile syndrome or Hurler-Scheie and Scheie for the juvenile and adult forms), MPS II (Hunter syndrome), MPS IIIA and MPS IIIB (Sanfilippo syndromes A and B). For MPS I and MPS II, the trials are based on intravenous AAV delivery of ZFN therapeutic agents to insert a functional copy of alpha- L-Iduronidase (IDUA) or iduronate 2-sulfatase (IDS), respectively, into the albumin locus of patient hepatocytes. These trails are currently being tested in adult patients aiming to improve the peripheral symptoms of these diseases. For MPS IIIA, the main goal is to deliver a functional copy of the human N-sulfoglucosamine sulfohydrolase (h.SGSH) directly into the brain of young children. Thus far, h.SGSH delivery seems to be safe, but the therapeutic benefit was limited with cognitive improvement observed in only one patient ( Vincent et al., 2014). For the treatment of MPS IIIB, the goal is to deliver N-sulfoglucosamine sulfohydrolase (NAGLU) to restore its expression in patients. Both intracerebral and intravenous delivery of AAV-NAGLU have shown to be promising in preclinical studies using rodent and dog models but whether this translates to therapeutic benefit in patients is yet to be demonstrated ( Ellinwood et al., 2011; Fu et al., 2011).

Pompe Disease

Pompe disease is a progressive neuromuscular disorder and is caused by a mutation in the acid alpha-glucosidase (GAA) gene, which encodes an enzyme required to degrade lysosomal glycogen ( Corti et al., 2015). Accumulation of glycogen in multiple tissues results in cardiac, respiratory, and skeletal muscle dysfunction. Preclinical studies in GAA knockout mouse models showed rescue of glycogen accumulation in muscle and the central nervous system, and increased survival upon delivery of GAA by AAV ( Puzzo et al., 2017). Delivery of GAA by AAV is currently being tested in three different clinical trials.

Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is caused by the loss of function of the gene encoding the survival motor neuron 1 (SMN1) protein and is characterized by progressive loss of the lower motor neurons. The most severe form is type 1 SMA which affect infants. Patients usually die before the age of 20 months. Several clinical trials are being conducted by AveXis based on AAV9-SMN delivery. Thus far, remarkable clinical benefit was observed including increased motor functions, head control, sitting, rolling over, and speaking. The high dose of intravenous injected AAV9-SMN led to an increase in serum aminotransferase, a sign of liver toxicity, which was managed by oral prednisolone. Intrathecal delivery of AAV9-SMN is also being investigated by AveXis as improved transduction of the CNS was observed at lower doses as compared to intravenous administration in NHP. This program is currently also being investigated in clinic for other types of SMA (NCT03306277, NCT03505099, NCT03461289, and NCT03381729), results of which are pending ( Table 4). A Biological License Application was submitted to the US FDA in November 2018 by Novartis, which acquired AveXis. In March 2019, Novartis announced the FDA approval of AAV9-SMS (Zolgensma®) for the treatment of pediatric SMA type 1 patients.

Huntington's Disease

Huntington's disease (HD) is the most common autosomal dominant neurodegenerative disorder worldwide ( Fisher and Hayden, 2014; Rawlins et al., 2016). The genetic cause is an expansion of 39 CAG triplets or more in first exon of the huntingtin (HTT) gene ( Miniarikova et al., 2018). The mutated gene produces a mutant HTT protein that contains a long polyglutamine (polyQ) tract and results in a toxic gain of function. The mutant HTT protein causes neuropathology affecting the entire brain, with medium spiny neurons of the striatum being particularly vulnerable at early stages ( Ross and Tabrizi, 2011; Bates et al., 2015). The clinical symptoms include progressive motor, cognitive, and psychiatric disturbances. Currently there is no treatment available that would halt or delay disease progression. Using artificial DNA or RNA molecules to achieve lowering of HTT translation as a potential therapy for HD has been broadly investigated in preclinical studies but few have made it to clinical trials. IONIS Pharmaceuticals was the first to initiate a Phase I/II clinical trial (NCT02519036) in 2015 based on HTT lowering with an intrathecally administered antisense oligonucleotide targeting both wild-type and mutant HTT. This trial was safe and a reduction of HTT protein was detected in the CSF ( Tabrizi et al., 2019). Roche is continuing this study with a large phase III trial (NCT03761849) that was launched in 2018 and will be conducted at around 80-90 sites in about 15 countries. It remains unknown whether this intrathecally delivered ASO will reach the deeper brain structures to achieve sufficient lowering of the mutant HTT in the striatum. Concerningly, the latest published data using this approach showed a dose- and time-dependent increase of the ventricular volume, which suggests that the treatment caused more CNS atrophy, instead of the desired prevention of CNS atrophy ( Tabrizi et al., 2019).

uniQure launched the first AAV-based RNAi therapy for HD in the second half of 2019 and the first two patients have been treated (ref. clinical trial: NCT04120493). The therapeutic candidates in AAV5-miHTT (AMT-130), which is anti-HTT miRNA targeting both wild-type and mutant HTT in a region close to the repeat in exon 1. AMT-130 was injected directly in the striatum of the brain and should provide a long-lasting production of miHTT. Due to the close proximity of miHTT target site to the CAG repeat, it is expected that AMT-130 can also target the short HTTexon1 mRNAs which are highly toxic. New preclinical data presented at the 15th Annual CHDI Huntington's disease Therapeutics Conference demonstrated that Q175FDN mouse model, treated with AMT-130 experienced a dose-dependent lowering of exon 1 HTT mRNA in both the striatum and cortex.

Safety and Delivery of Gene Therapy to Patients

The use of AAV vectors as transgene delivery has become one of the safest and most reliable methods to obtain sustained expression of a therapeutic transgene. Non-viral delivery methods using nanoparticles or ASOs can also deliver therapeutic agents but the need for recurrent injections can become overwhelming and increase the risk for infections in patients. Nevertheless, ASOs have shown to be efficient and safe in animal models and have led to the initiation of several clinical trials for SOD1-ALS, C9orf72-ALS, and SMN2 for SMA ( Miller T. et al., 2013; Meyer et al., 2015; Mendell et al., 2017).

The delivery of gene therapy to the affected cells in patients suffering from neurodegenerative diseases is currently a major challenge. Several studies have implicated that the utility of AAV vectors for gene transfer is mainly determined by the capsid. We believe that this statement is partly true as the capsid is involved in several key cell entry steps. However, the route of administration is equally important as capsids behave differently upon different administration routes due to different microenvironments ( Pietersz et al., 2020). For example, it was demonstrated that intraparenchymal injection of AAV5 resulted in high transduction of the rat brain ( Pietersz et al., 2020). Similar findings were observed in a transgenic minipig model showing successful transduction at the injection site (striatum) and its surrounding area ( Evers et al., 2018). In addition, both anterograde (e.g., to caudate) and retrograde (to cortex) AAV5 viral transport was observed, suggesting that the vector can travel along axons and transduce distant areas. While this route is promising for diseases with a more localized pathology, such as HD, it is unlikely to be sufficient in multifocal neurological pathologies such as ALS because anterograde and retrograde transport to cerebellum, brain stem, and spinal cord was not observed in this study. In Rats, intrathecal administration led to a more even distribution of the vector within the CNS and is potentially more relevant for diseases with multifocal neurological pathologies ( Pietersz et al., 2020). However, it remains a challenge to deliver a therapeutically relevant dose throughout the entire CNS in larger animals. Another crucial aspect is safety, especially due to the irreversible nature of current gene therapies, evaluation of short term and long-term toxicity is critical. Other crucial parameters that needs to be addressed during preclinical studies are prediction of on- and off-target effects, immunogenicity, dose finding, timing of treatment, and the ability to modulate gene expression.

Prediction of Safety, On- and Off-Target Effect

The most important aspect for drug development is safety and tolerability in patients. Both transgene products and delivery vector could lead to toxicity. Several clinical studies have demonstrated that administration of AAV in human CNS is safe, but immune responses should still be investigated during preclinical and clinical studies. The transgene products that are delivered could also induce on- and off-target effects, for example in the case of RNAi-based gene therapy. Prediction of these unwanted side effects can be evaluated early in preclinical studies using the currently available tools such as iPSC-technology, animal models, and bioinformatics to increase safety and tolerability in patients.

Timing of Treatment

The success of a gene therapy is likely to be determined by the timing. Most of the adult onset neurodegenerative diseases have a pre-symptomatic stage and it may take several years to gradually progress into a severe state with progressive neuronal death. While neuronal death cannot be reversed, emerging evidence suggests that neurons in atrophic state can regenerate their normal functions ( John and Petitto, 2014). Thus, early treatment before neuron death occurs is likely to provide better protection and could decelerate neuronal death. Currently all gene therapy clinical trials are performed during the manifest state in patients while treatment during pre-manifest could be more beneficial. With the current knowledge and technologies, neurodegenerative diseases with a family history can be diagnosed prenatally or later in life during the pre-symptomatic phase using genetic screening. The discovery and characterization of more clinically validated biomarkers that correlate with disease progression will also contribute to early diagnosis of both familial and sporadic neurodegenerative diseases. Biomarkers that track disease progression and correlate predictably in response to a therapeutic intervention can also greatly support future clinical trials by reducing the duration of the studies and number of patients that need to be followed. This is especially important in pre-manifest subjects when no clinical measures of disease progression can be applied. However, due to the lack of effective treatment options diagnoses during the premanifest stage also raises many ethical concerns and even patients at a high risk of developing these diseases are fearful to do a genetic screening. Patients who test positive are currently faced with difficult decisions that can have mayor psychological and financial impacts on the patients themselves and their families. For example, a positive genetic test could have implications on the daily life of a person, their health insurance, their ability to find a job, and on decision making with may affect their family ( Cozaru et al., 2016; Uhlmann and Roberts, 2018). Ultimately, a positive genetic test may have consequences regarding a person's ability to get a mortgage and integrate into society ( Wauters and Van Hoyweghen, 2018). This is a phenomenon known as " genetic discrimination " and needs further attention. The success of new therapeutics will hopefully extend to trials in premanifest carriers and could make it easier for patients at risk to make the decision to do a genetic screening.

Modulation of Gene Expression

Besides constitutively active promoters to drive transgene expression, the ability to "turn on" expression of a therapeutic molecule when it is needed and to "turn off" its expression in case of unwanted effects would also add considerably to the safety profile of any genetic therapy. The feasibility of different inducible transgene expression systems to use in combination with gene therapy has been demonstrated in animal studies but translation to the clinic is lacking ( Cheng et al., 2018; Liefhebber et al., 2019). The incorporation of the inducible systems to regulate gene therapy would add to the safety by making adjustments on individual basis possible and preventing too high concentrations of transgene products that could be detrimental. However, there is still room for improvement to make inducible systems suitable for clinical applications. One drawback is the basal activity of these systems in the "off-state." Especially small transgenes such as non-coding RNAs tend to be leaky when using inducible systems and further optimizations are needed. Another aspect that needs more attention isobtaining of sufficient transgene expression once in the on-state. Especially for CNS diseases, the inducer drug needs to efficiently cross the BBB and reach the target tissue at concentrations that are high enough for therapeutically relevant expression of the transgene. Possible immune responses to elements inherent to the currently available inducible systems and side effects of the inducer drug should also be considered for clinical development. The co-development of two different products (inducible system components and transgene) in either a single or duo vector also add more parameters to the safety concerns of gene therapies, but these systems also offer notable advantages. Being able to modulate transgene expression will at least reduce safety concerns of the permanency expressed transgene. In turn making further investigation and optimization of these systems highly attractive for application in gene therapy.

Future Perspectives

Gene therapy holds great promise for delivering therapeutic genes to treat neurological disorders. AAV vectors are currently considered one of the safest vehicles to treat CNS disorders. Several serotypes are available and as the potential of gene therapy is increasingly recognized, there is emerging need for new AAV serotypes. There is an enormous desire for new AAV vectors with improved transduction profile, better distribution, and higher transduction in the target organs upon less invasive administration routes. The genetic modification of AAV vectors and engineering AAV vectors could overcome these hurdles. The selection of appropriate delivery routes is also an important factor for the success of gene therapy. Currently, direct administration of AAV vectors into the parenchyma is preferred for efficient transduction of the CNS. Although this route of administration is invasive, the advantages over injections into the venous system or other fluid-filled compartments are clear. Intra-parenchymal administration provides high concentration of the transgene in the target cells, high local transduction, less distribution to other organs, and lower risk for immune responses or toxicities due to AAV particles or ectopic expression of the transgene. Administration of AAV via systemic or intrathecal routes require higher doses which in turn increase the risk for toxicity. There is also a lot of room for improvement at the transgene level. For example, generation of new promoters and cis- elements, optimization of codons, and more efficient transgene design could improve efficacy and restrict expression in specific CNS cell types.

Throughout this review several clinical trials on AAV-based gene therapies for CNS disorders have been discussed ( Piguet et al., 2017). Although all studies showed that AAV is safe and tolerated well in the human CNS, few of these studies have been efficacious in demonstrating therapeutic benefit. Too low transduction of the target organs could have played an important role, but one major problem is the lack of good predictive preclinical models for a more accurately translation of favorable preclinical outcomes to the clinic. Many regions of the CNS are difficult to access after in vivo delivery of viral vectors, resulting in a reduced number of cells expressing the therapeutic transgene which may be insufficient to reach overall required therapeutic levels ( Piguet et al., 2017). Furthermore, the discovery of biomarkers for early detection of the diseases will be important to identify new patients and to better predict clinical outcome. More importantly, delivering therapies prior to onset of neurodegeneration will be key to improving efficacy of gene therapies for neurodegenerative diseases. A better understanding of factors leading to these diseases will also facilitate development of better pre-clinical models that make it possible to better predict clinical success. Results from clinical trials are raising and the successes and lessons learned from the past and current AAV gene therapy clinical trials will be highly valuable for application to a wider range of neurodegenerative and neuromuscular diseases.

Author Contributions

Both authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

Conflict of Interest

RM and PK were employed by the uniQure Biopharma B.V.

Footnotes

References

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Gene Therapy for Neurodegenerative Diseases: Slowing Down the Ticking Clock

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Gene therapy is an emerging and powerful therapeutic tool to deliver functional genetic material to cells in order to correct a defective gene. During the past decades, several studies have demonstrated the potential of AAV-based gene therapies for the treatment of neurodegenerative diseases.

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Main Difference - Lentivirus vs Retrovirus

Lentivirus and retrovirus are two types of viruses that infect living cells for the completion of their life cycle. Lentivirus is a subtype of retroviruses. The main difference between lentivirus and retrovirus is that lentivirus can infect both actively-dividing and non-dividing cells whereas retroviruses can only infect mitotically-active cell types. Thus, lentivirus can infect a wide range of cell stages than retroviruses. Both lentivirus and retrovirus can be used as vehicles that carry foreign DNA molecules into another cell. The mechanism of DNA transfer mediated by viruses is known as transduction. Both lentiviruses and retroviruses are used in to correct defective genes that are responsible for disease development.

Key Areas Covered

1. What is a Lentivirus
- Definition, Properties, Types
2. What is a Retrovirus
- Definition, Properties, Types
3. What are the Similarities Between Lentivirus and Retrovirus
- Outline of Common Features
4. What is the Difference Between Lentivirus and Retrovirus
- Comparison of Key Differences
Key Terms: HIV, Lentivirus, Recombinant DNA Technology, Retrovirus
What is Lentivirus

Lentivirus is a type of retrovirus that consists of long incubation periods and cause chronic, progressive, and typically fatal diseases in animals. Lentiviruses can be subdivided into five serotypes based on the type of vertebrate host they infect; bovine, equine, feline, ovine/caprine, and primate. The human immunodeficiency viruses (HIV) type and 2, simian (SIV), and feline (FLV) are examples of lentiviruses. Lentiviruses are used to introduce large DNA segments into the cell line (dividing), primary cells (dividing and non-dividing), neuronal cells, stem cells, and hematopoietic cells. Most lentiviral vectors are based on HIV. The structure of HIV is shown in figure 1.

The size of the HIV genome is 9.7 kb. The three major structural genes present in the genome are the gag (group-specif antigen), pol (polymerase), and env (envelope). The other regulatory genes are tat (HIV transactivator) and rev (regulator of expression of virion protein). In addition, four types of accessory genes are also present in the HIV genome: vif (viral infectivity), vpr (virus protein R), vpu (virus protein U), and nef (negative factor). Replication, integration, and packaging of lentiviruses are mediated by cis-acting elements of the lentiviral genome.

What is Retrovirus

Retrovirus is any type of RNA viruses that insert a DNA copy into the host genome for replication. Retroviruses are characterized by their ability to reverse transcribe their RNA genome into a cDNA copy that can be integrated into the host genome. They consist of two copies of positive sense ssRNA molecules in the genome. Retroviruses can be classified into three subfamilies based on the different types of accessory and regulatory genes present in the genome. They are oncoviruses, lentiviruses, and spumaviruses. Oncoviruses are subclassified into three categories based on the morphology; B-type, C-type, and D-type. The different subfamilies of retroviruses with examples are described in table 1.

Retrovirus Subfamilies and Examples

Oncovirus B-type

Murine mammary tumor virus (MMTV)

Oncovirus C-type

Human T-cell leukaemia virus (HTLV), Avian leukosis and sarcoma viruses (ALSV), and Salmon lymphoma virus

Oncovirus D-type

Mason-Pfizer monkey virus (MPMV)

Lentiviruses

Human immunodeficiency viruses (HIV), Ovine maedi-visna virus (MVV), and Equine infectious anaemia virus (EIAV)

Retroviruses consist of reverse transcriptase enzyme for the reverse transcription of the RNA genome. Structural and enzymatic proteins such as capsid (CA), nucleocapsid (NC), integrase (IN), and protease (PR) are located inside the retroviral compartment.

The inner core of the retrovirus is surrounded by an outer protein layer, consisting of the matrix (MA) protein. The envelope of the retrovirus is derived from the host cell membrane. Retroviruses infect dividing cell lines and primary cells, stem cells, and hematopoietic cells.

Similarities Between Lentivirus and Retrovirus

Lentivirus and retrovirus are two types of viruses that infect living cells.

The capsid of both lentivirus and retrovirus is icosahedral in shape and both are enveloped viruses.

Both lentivirus and retrovirus consist of two copies of positive sense ssRNA genome.

Both lentivirus and retrovirus consist of reverse transcriptase (RT).

Both lentivirus and retrovirus are capable of reverse transcribing their RNA genome into a cDNA copy.

The genome of both lentivirus and retrovirus are encoded for polymerases.

Both lentivirus and retrovirus can be used as vehicles that carry foreign DNA molecules into another cell.

Both lentivirus and retrovirus are used in recombinant DNA technology to introduce comparatively large foreign DNA segments into animal cells.

Both lentiviruses and retroviruses are used in gene therapy to correct defective genes that are responsible for disease development.

Both lentivirus and retrovirus use different isoforms of gag, pol, and env genes for packaging.

Both lentivirus and retrovirus can infect dividing cells, stem cells, and hematopoietic cells.

Both lentivirus and retrovirus have a broad range of host range.

The size of the insert of both lentivirus and retrovirus is around 8.0 kb.

Both lentivirus and retrovirus DNA are integrated into the genome.

Both lentivirus and retrovirus can be integrated up to 10 copies.

Both lentivirus and retrovirus do not have a very high level of protein expression.

The recommended biosafety level of both lentivirus and retrovirus is BSL-2.

Difference Between Lentivirus and Retrovirus

Definition

Lentivirus: Lentivirus is a type of retrovirus, consisting of long incubation periods and causing chronic, progressive, and typically fatal diseases in animals.

Retrovirus: Retrovirus is any type of RNA virus that inserts a DNA copy into the host genome for replication.

Types of Infecting Cells

Lentivirus: Lentivirus can infect both actively-dividing and non-dividing cells.

Retrovirus: Retroviruses can only infect mitotically-active cell types.

Types

Lentivirus: Lentivirus is a type of retroviruses.

Retrovirus: The three types of retroviruses are oncoviruses, lentiviruses, and spumaviruses.

Range

Lentivirus: Lentivirus can infect a wide range of cell stages.

Retrovirus: Retrovirus can infect a narrow range of cell stages.

Size of the Genome

Lentivirus: The size of the wild-type lentiviral genome is 9.7 kb.

Retrovirus: The size of the wild-type retroviral genome is 8.3 kb.

Accessory Genes

Lentivirus: Lentiviruses consist of accessory genes in their genome.

Retrovirus: Some retroviruses such as oncoviruses lack accessory genes.

Conclusion

Lentivirus and retrovirus are two types of viruses that infect living cells. Lentivirus is a type of retroviruses. Both lentivirus and retrovirus are used in recombinant DNA technology to introduce comparatively large foreign DNA segments into animal cells. Lentivirus can infect both dividing and non-dividing cells but, retroviruses can infect only dividing cells. This is the main difference between lentivirus and retrovirus.

Reference:

1. O'Keefe, Eric P. "Nucleic Acid Delivery: Lentiviral and Retroviral Vectors." Materials and Methods, 15 June 2015, Available here.

Image Courtesy:

1. " HI-Virion-en" By US National Institute of Health (redrawn by en:User:Carl Henderson) - US National Institute of Health (redrawn by en:User:Carl Henderson) (Public Domain) via Commons Wikimedia
2. "Hiv gross" By Translated by Raul654 - Originally from GFDL image Image:Hiv gross german.png (CC BY-SA 3.0) via Commons Wikimedia

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Difference Between Lentivirus and Retrovirus | Definition, Properties, Types, Similarities and Differences

# data[].attributes.attachment_link.description
Lentivirus and retrovirus are two types of viruses that infect living cells for the completion of their life cycle. Lentivirus is a subtype of retroviruses. The main difference between lentivirus and retrovirus is that lentivirus can infect both actively-dividing and non-dividing cells whereas retroviruses can only infect mitotically-active cell types.

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The past year revealed both successes and setbacks for viral-vector gene therapies. The rapid development and large-scale rollout of multiple adenovirus-vector vaccines represented an unprecedented achievement that is poised to help mitigate the devastating impact of the COVID-19 pandemic. During the same period, multiple high-profile gene-therapy assets encountered challenges, with clinical trials paused because of safety concerns or failing to meet efficacy targets.

These successes and setbacks are emblematic of the current state of viral-vector gene therapy: a technology with considerable promise but with a set of challenges still ahead. As more and more gene therapies have reached the clinic, it has become clear that multiple technological challenges must still be overcome to unlock the full potential of viral-vector gene therapy.

Rising to meet these challenges, biotech and pharmaceutical companies are testing a multitude of technological advances and innovative strategies that address all aspects of viral-vector gene-therapy development. For companies prepared to keep abreast of the rapid pace of change, these innovations offer a path for ushering in the next generation of viral-vector gene therapies.

Companies are testing a multitude of technological advances and innovative strategies that address all aspects of viral-vector gene-therapy development.

The state of viral-vector gene therapy

Viral-vector gene therapies use modified viruses as drug-delivery vehicles to introduce specific DNA sequences-encoding genes, regulatory RNAs (for example, small interfering RNAs [siRNAs]), or other therapeutic substrates-into cells. The technology has long drawn interest for its potential advantages over traditional modalities. Many types of therapeutic agents (for example, enzymes, antibodies, and siRNAs) can be encoded in DNA sequences that can be rapidly designed and synthesized once a target is identified.

Viruses serve as powerful delivery vehicles for these sequences because of their ability to enter cells efficiently and potentially gain access to hard-to-reach, highly specific cells. In combining these features, viral-vector gene therapies can be used to modify gene expression in a programmable way, offering the flexibility to potentially treat a wide spectrum of diseases-including rare monogenic diseases by gene replacement and broad-population diseases by controlling gene expression-and help disease prevention by immunization.

Nearly all gene therapies currently available use one of three vector types: adeno-associated-virus (AAV) vectors, adenovirus vectors, or lentivirus vectors (Exhibit 1). AAV and adenovirus vectors are typically used in gene therapies that are directly administered to patients by infusion or local administration ( in vivo), with AAV being the most popular vector for areas outside of oncology and vaccines. Lentivirus vectors are typically used for ex vivo therapies, in which cells harvested from a patient are modified in the lab before retransplantation. This article primarily focuses on in vivo gene therapies; however, many of the challenges and advances discussed are applicable across both routes of administration.

Excitement around viral-vector gene therapies is evident. While only four in vivo viral-vector gene therapies are currently on the market, more than 100 gene-therapy assets are in clinical trials as of late 2020, with a far greater number in preclinical development.

However, while there is significant momentum, there have also been multiple recent setbacks. Many of these relate to challenges previously outlined by McKinsey in its perspective on the future of gene therapy (including efficacy, durability, and manufacturing). As these therapies have sought to expand beyond the ultrarare indications they originally targeted, three technological challenges have emerged as recurrent obstacles. For viral-vector gene therapies to reach their true transformative potential-much like monoclonal-antibody technology 20 years ago-this set of technological challenges must be overcome.

Challenges to realizing the potential of viral-vector gene therapies

The current generation of viral-vector gene therapies represents the culmination of decades of biological and clinical research. As more patients have received these therapies, it has become clear that three fundamental challenges will restrict the applicability of viral vectors: getting past the immune system, lowering the dose, and controlling transgene expression. Ongoing work to address these challenges is generating technological innovations that have the potential to leapfrog current therapies and unlock the potential of viral vectors.

Getting past the immune system

The success of any viral-vector gene therapy depends on its ability to get past multiple lines of defense deployed by the human immune system. Viral capsids, viral-vector DNA, and even the transgene products themselves may be recognized as foreign, providing multiple opportunities for the immune system to clear the gene therapy from the body.

Although this effect depends on the vector serotype used, and the clinical impact is still unclear, many clinical-trial sponsors conservatively exclude patients from their studies if they have antibodies to the vector in question. This can come at the cost of making most patients ineligible for therapy. Acquired immunity to viral vectors poses additional challenges for viral-vector gene therapy in the long term. Patients treated with a gene therapy today may not be able to receive a second gene therapy in the future if the same viral vector is used in both contexts.

In addition, viral capsids and viral-vector DNA can actively provoke an immune response from the body. For viral-vector vaccines, this immunogenicity can be beneficial, as it reduces the need for adjuvants and increases efficacy. However, for other viral-vector gene therapies, immunogenicity can reduce efficacy, increasing the chance that the gene therapy is detected and eliminated by the immune system. Indeed, some have speculated that immunogenic vector DNA sequences are behind the limited durability of some recent gene therapies, leading to their abandonment. More concerningly, immunogenicity can lead to safety concerns during therapeutic use, as high levels of viral capsids can cause severe immune reactions at the time of injection.

Unraveling the immune system's intertwined responses to viral-vector gene therapies remains difficult. Animal models do not recapitulate all relevant aspects of the human immune system (as immune systems behave quite differently among species). While human clinical trials offer a valuable source of insight, many gene-therapy trials are too small to confidently isolate the parameters associated with a drug's success or failure.

Lowering the dose

The large doses used in current gene therapies pose two challenges. First, large doses are difficult and expensive to manufacture. Today, a typical manufacturing run of an AAV-vector therapy using high-yield cell lines and large-capacity bioreactors might only produce approximately ten doses of a systemic gene therapy from a single batch at a cost of nearly $100,000 per dose (assuming approximately 1 × 10^17 vg per batch). Although these costs will gradually decrease as gene therapies begin to reach clinical and commercial scales, any technological advance that reduces the required dose would bring immediate benefit, as a tenfold reduction in dose might also bring about a tenfold reduction in costs.

Controlling transgene expression

Once a viral vector successfully delivers its therapeutic gene to the cells in question, the efficacy of the gene therapy depends on the quality of transgene expression. Specifically, the transgene must be expressed at the appropriate level (neither too low nor too high), in the appropriate cells, and for the appropriate duration to mediate the desired clinical effect. For therapeutic uses (in contrast to use for vaccines), the transgene may need to be expressed permanently if the gene therapy is to serve as a one-time cure and represent an appealing alternative for patients over current standards of care requiring repeated dosing (which may not be possible because of the challenges previously laid out). Regulators have required multiple years of follow-up data showing that gene expression is maintained. Indeed, some drugs have been abandoned when expression waned after 12 months.

To maximize chances of success, early viral-vector gene therapies have opted to include regulatory elements (DNA sequences such as promoters and enhancers that control how genes are expressed) that have been selected to drive high levels of transgene expression in all cell types. However, this approach may have significant drawbacks, particularly as gene therapies move beyond gene replacement for monogenic rare diseases. Overexpression of the transgene or its expression in the wrong cells may contribute to inflammation and other toxicities (as was observed in recent studies of nonhuman primates). Moreover, current gene therapies, once administered, cannot be controlled or turned off by clinicians should the need ever arise.

Innovative solutions that address gene-therapy challenges from many angles

To tackle the challenges facing gene therapy, academic labs, start-ups, and established companies are generating myriad innovative solutions (Exhibit 3). Each focuses on a specific component of a gene-therapy product (for example, the viral capsid) or part of the development process (such as manufacturing). However, these innovations often address multiple core challenges, outlining multiple paths to realizing the promise of viral-vector gene therapy.

We have identified five key trends to watch.

1. Improved capsids

The viral capsid is a critical component of viral-vector gene therapy. It determines which cells are targeted, the efficiency of cell entry, and the probability that the gene therapy is detected and eliminated by the immune system. In addition, the capsid is largely responsible for the stability of the viral vector during the manufacturing process and can affect storage and distribution requirements.

The capsids most widely used today, including those used in on-market products, are derived from naturally occurring viruses. They have suboptimal properties, including little cell-type specificity, moderate efficiency of cell entry, and relatively high levels of preexisting immunity in humans. To address the problem of preexisting immunity, many assets use capsids from viruses found in other species. For example, the AAV8 and AAVrh74 capsids used in multiple AAV-vector gene therapies are derived from AAV serotypes isolated from macaques, and some of the COVID-19 vaccines that have been developed have used adenovirus serotypes from chimpanzees and gorillas. While this approach may limit the challenges of preexisting immunity, it largely doesn't address specificity or efficiency (particularly as these viruses have evolved to infect nonhuman species).

Increasingly, drug developers are turning to capsids that have been engineered in the lab and can be selected to overcome the challenges mentioned previously (Exhibit 4). These engineered capsids are identified through large-scale screening efforts in which millions of variant capsids are screened for the desired properties and iteratively refined. Capsid-engineering platforms-many of which have been spun out of academic labs to form companies-achieve these ends by leveraging advanced technologies, such as cryo-electron microscopy (cryo-EM) and artificial intelligence.

Improving capsid properties could bring multiple immediate benefits. For example, a twofold increase in a capsid's cell-type specificity could enable a twofold decrease in the overall viral dose required, thereby improving safety and cost. It's still too early to determine the true impact of capsid engineering, as most engineered capsids are still in preclinical development. However, companies' early reports suggest that capsids with five- to tenfold improvements in multiple attributes may be entering the clinic soon.

2. Improved vectors

Like the capsid, the DNA sequence of the viral vector itself affects multiple aspects of a gene therapy's performance, but engineering the vector can often be considerably easier, cheaper, and quicker. Accordingly, vector engineering is becoming a growing focus of gene-therapy R&D. Vector engineering is often easier with adenovirus and lentivirus vectors than with AAV vectors because of AAV's inability to package large pieces of DNA. However, innovative vector elements are beginning to appear in AAV-vector designs as well.

Vector engineering broadly has two aims: reducing the immunogenicity of the viral vector and improving transgene expression. One strategy to achieve both aims is codon optimization, in which variations in the vector sequence are explored to eliminate immunogenic sequence motifs while optimizing the transgene for robust expression. Subtle changes in vector sequence achieved through codon optimization can have large effects, such as increasing expression levels and possibly extending the duration of expression for multiple years.

Vector engineering broadly has two aims: reducing the immunogenicity of the viral vector and improving transgene expression.

Transgene expression can be further programmed by engineering regulatory elements into the vector sequence. Some regulatory elements turn on transgene expression only in certain cell types or tissues-ideally, the disease-causing cells-preventing potentially toxic expression in other contexts. Such cell-type- or tissue-specific regulatory elements (for example, promoters and enhancers) have become relatively common in viral-vector gene therapies. For an additional layer of control, some viral-vector gene therapies are also incorporating regulatory elements, such as microRNA-target sites, that reduce expression in specified cells-for example, in cells that promote an immune response.

Finally, a more distant and challenging goal is to engineer vectors that are inducible, where transgene expression can be controlled using an additional signal, such as an orally administered small-molecule drug. This could allow clinicians to turn on, turn off, or otherwise adjust a gene therapy after it is administered, delivering a personalized course of treatment.

3. New types of cargo

The cargo delivered by a viral-vector gene therapy is typically a working copy of a gene that is used to replace the patient's disease-causing copy of that same gene. However, any therapeutic agent that can be encoded in DNA can theoretically be delivered by a viral vector. Researchers and drug developers are increasingly leveraging this flexibility to deliver other types of molecules with therapeutic value-alone or sometimes in combination-including regulatory RNAs (for example, short hairpin RNAs [shRNAs]), vectorized antibodies, and substrates for gene editing.

Gene editing is an intriguing potential solution for achieving long-lasting, physiologically appropriate gene expression. For patients with diseases caused by certain types of mutations, restoring the function and expression of the patient's own copy of the gene through gene editing may be simpler (and more permanent) than attempting to engineer and deliver a replacement.

4. Improved manufacturing processes

Early gene-therapy-manufacturing processes originated in academic labs and were focused on small, research-scale batches. These processes were not optimized for moderate- or large-scale production or for the delivery of systemic therapy. As gene therapies start to expand outside the treatment of ultrarare diseases, one of the many challenges being addressed is the presence of empty capsids created during the manufacturing process. These empty capsids, which have no active cargo, can create the requirement for higher doses and, accordingly, stimulate stronger immune responses.

Two approaches are being developed to reduce the ratio of empty-to-full capsids in manufacturing: developing improved methods to separate the empty from full capsids based on specific properties (for example, charge and molecular weight) and engineering cell lines that package full capsids more efficiently. By reducing the empty-to-full ratio, these advances reduce manufacturing costs, reduce immune responses, and improve the safety of gene therapy. Indeed, regulators have used reducing the empty-to-full capsid ratio as part of the rationale for lifting clinical holds on gene-therapy products with previous safety issues.

5. Improved pretreatment and conditioning regimens

Beyond engineering the capsid and vector, a separate approach for reducing the immune system's detection of viral-vector gene therapies involves coadministering the therapy with an immunosuppressive agent. Multiple such conditioning regimens are currently being tested to reduce the impact of neutralizing antibodies on the efficacy of the treatment, both of preexisting antibodies and newly generated antibodies that could prevent future redosing. Nearly all current viral-vector gene therapies use steroids to help manage the potential immune response to the viral vector; however, the type, dosage, and timing of the steroid treatment varies widely.

The road ahead

Viral-vector gene therapies find themselves at another inflection point. Early successes in the treatment of rare diseases and vaccines have proven the potential of this modality, while the challenges to gaining widespread adoption-the way that monoclonal antibodies have over the past 20 years-have only become clearer. Nevertheless, the wealth of innovative solutions being explored across academia, biotech, pharma, and contract development and manufacturing organizations demonstrate that viral-vector gene therapies are here to stay.

As described previously, different solutions are emerging to address each of the core challenges. The diversity of these approaches and the complexities of gene therapy mean that no single approach is likely to "win." That situation will enable a rapid innovation cycle in which gene therapies are constantly being improved upon, which will offer new opportunities to leapfrog existing products. Even as AAV-vector-based delivery is becoming the leading technology, some prominent limitations combined with the rapid pace of innovation leave the door open for other delivery technologies to emerge.

Owners of viral-vector platforms will need to consistently look to the next set of innovations beyond their current platforms and assets. That could include investing directly to help overcome the broader challenges or buying or licensing critical technology to upgrade their platforms. Indeed, multiple new biotech companies have launched to solve one or more of the challenges outlined in this article as a service to developers of gene therapies. Staying abreast of these developments will require fastidious monitoring of scientific and technological progress on all fronts. However, since it is difficult at this early stage to place bets across all potential solutions and innovators, gene-therapy leaders will need to make their investments judiciously.

In the short to medium term-while technological challenges limit the scope of gene therapies to curative treatments for rare diseases-fast followers may find it difficult to be successful, even with improved technologies, as first entrants rapidly address prevalent populations. Gene-therapy leaders will therefore need to strike a careful balance by accelerating programs today while retaining the flexibility to adopt innovative technologies that unlock treatments for broader-population diseases and the full promise of viral-vector gene therapies in the long term.

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Gene-therapy innovation: Unlocking the promise of viral vectors

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The past year revealed both successes and setbacks for viral-vector gene therapies. The rapid development and large-scale rollout of multiple adenovirus-vector vaccines represented an unprecedented achievement that is poised to help mitigate the devastating impact of the COVID-19 pandemic.

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Gatsby Plant Science Education Programme

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Making new vaccines from plant viruses - George Lomonossoff

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Viruses: Infectious Medicine | Bertram Jacobs | TEDxASU

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Cassava brown streak viruses (cassava brown streak disease) 

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Cassava brown streak viruses (cassava brown streak disease) | CABI Compendium

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This datasheet on Cassava brown streak viruses covers Identity, Overview, Distribution,
Dispersal, Hosts/Species Affected, Vectors & Intermediate Hosts, Diagnosis, Biology
& Ecology, Seedborne Aspects, Impacts, Prevention/Control, Further ...

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Viral Vectors for Plant Genome Engineering

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Recent advances in genome engineering (GE) has made it possible to precisely alter DNA sequences in plant cells, providing specifically engineered plants with traits of interest. Gene targeting efficiency depends on the delivery-method of both sequence-specific nucleases and repair templates, to pla…

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When the green revolution began in the 1960s, it was before the revolution in molecular genetics: IR8, the first miracle rice, was bred without knowledge of the genes that blessed it with high yields. Breeders today can zero in on genes, but they still use traditional techniques and ever more complex pedigrees. That's how they've created rice varieties adapted to rising sea levels-including Swarna-Sub1, popular in India, and IR64 Sub1, whose pedigree is shown here.

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Can science prevent our next food crisis?

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What Is the Green Revolution? | World History Project

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Virus DNA in human genome (evolution by infection)

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The story of water will play out in front of a new satellite mission launching tomorrow (Dec. 15).

The Surface Water and Ocean Topography (SWOT) satellite aims to map Earth's water in unprecedented detail to help manage climate change, and to share data openly to assist communities with managing the precious resource. It launches aboard a SpaceX Falcon 9 rocket no earlier than 6:46 a.m. EST (1146 GMT) and we will run coverage here at Space.com.

"Let me tell you, SWOT is a game changer," Tahani Amer, a program executive in NASA's Earth science division, said during a livestreamed Nov. 14 NASA science briefing. Amer added that SWOT will work closely with other satellites to provide valuable context about water, while other missions focus on areas like the atmosphere or ice cover.
Related: 10 devastating signs of climate change satellites can see from space
NASA and France's space agency (CNES) are jointly leading the multi-agency effort. One crucial application will be better understanding carbon dioxide, a greenhouse gas, and its role in climate change, Katherine Calvin, NASA's chief scientist and senior climate advisor, said in a livestreamed Tuesday (Dec. 13) NASA briefing.

"Some of that carbon goes into the ocean, some goes onto land absorbed by trees, other [carbon] stays in the atmosphere," Calvin explained. "As it [Earth] gets warmer, the oceans are absorbing a lot of that heat. So a better understanding that mixing process of the ocean will help us understand how much more heat and carbon we can uptake. That's really important for understanding future climate change, and how activities by humans influence future climate change."

What makes SWOT distinct from water missions before it is three things: its global reach (which will especially help with assessing freshwater resources and ocean coastlines), its ability to detect small-scale but crucial ocean drivers at a resolution of less than 100 kilometers (62 miles), and the applications and data that will flow from the mission.

SWOT has a new measurement system on board named KaRIn, which stands for Ka-band Radar Interferometer. Ka-band is a part of the microwave spectrum of light and can collect data at high resolution, thanks to twin antennas positioned roughly 33 feet (10 meters) apart - about half the length of a tennis court, NASA officials say.

KaRIn sends radar pulses from one of the antennas that reflect off the surface of the Earth, and then both antennas work together to pick up the signal. The antennas' joint efforts allow scientists to map the surface water in two dimensions, as long as some key parameters are met.

At all times, scientists will need to know exactly how high the satellite is flying above Earth (which is an approximate 500 miles or 890 km). Precise positioning is provided through DORIS, the Doppler Orbitography and Radiopositioning Integrated by Satellite instrument. DORIS will do its job by collecting signals from as many as 60 radio beacons on the ground.

Also on board are an X-band radio communications system to send the data back to scientists, a radiometer to correct for the delay in receiving signals from moving water, a traditional altimeter, and a thermal management system to keep the satellite stable in the extreme heat and cold of space.

(Image credit: NASA)

In photos: Iconic images of Earth from space

From the Congo Basin to the north of Canada where Indigenous populations live, SWOT aims to provide community-focused solutions to climate change through the open data.

"SWOT is going to provide really relevant information for all these communities, all these people living in these different places and allow us to make measurements that ultimately improve their lives and livelihoods," Benjamin Hamlington, research scientist in the sea level and ice group at NASA's Jet Propulsion Laboratory, said in a Dec. 13 briefing.

Climate change is causing different effects across the world, with some people seeing far too little water and some seeing far too much, he said. Large rivers such as the Connecticut River, which flows across several northeastern states in the U.S., will see their full water levels measured simultaneously along with any changes.

Hamlington emphasized communities won't be on their own with the data, as expertise is available in the form of water resource managers, emergency preparedness agencies and civil engineers.

"We're not just providing the data but encouraging people to use it, interact with it and start to implement it within their applications," he added, with "early adopters" including communities in India and Germany who will help NASA better adjust data gathering when the mission finishes its months-long commissioning phase in 2023.

Elizabeth Howell is the co-author of "Why Am I Taller (opens in new tab) ?" (ECW Press, 2022; with Canadian astronaut Dave Williams), a book about space medicine. Follow her on Twitter @howellspace (opens in new tab) . Follow us on Twitter @Spacedotcom (opens in new tab) or (opens in new tab) .

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NASA's new SWOT satellite to study Earth's water from space in a 'game changer' for understanding climate change

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The story of water will play out in front of a new satellite mission launching tomorrow (Dec. 15). The Surface Water and Ocean Topography (SWOT) satellite aims to map Earth's water in unprecedented detail to help manage climate change, and to share data openly to assist communities with managing the precious resource.

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The urgent need to remove excess carbon dioxide from Earth's environment could include enlisting some of our planet's smallest inhabitants, according to an international research team led by Michael Hochella of the Department of Energy's Pacific Northwest National Laboratory.

Hochella and his colleagues examined the scientific evidence for seeding the oceans with iron-rich engineered fertilizer particles near ocean plankton. The goal would be to feed phytoplankton, microscopic plants that are a key part of the ocean ecosystem, to encourage growth and carbon dioxide (CO ₂) uptake. The analysis article appears in the journal Nature Nanotechnology.

"The idea is to augment existing processes," said Hochella, a Laboratory fellow at Pacific Northwest National Laboratory. "Humans have fertilized the land to grow crops for centuries. We can learn to fertilize the oceans responsibly."

In nature, nutrients from the land reach oceans through rivers and blowing dust to fertilize plankton. The research team proposes moving this natural process one step further to help remove excess CO ₂ through the ocean. They studied evidence that suggests adding specific combinations of carefully engineered materials could effectively fertilize the oceans, encouraging phytoplankton to act as a carbon sink. The organisms would take up carbon in large quantities. Then, as they die, they would sink deep into the ocean, taking the excess carbon with them. Scientists say this proposed fertilization would simply speed up a natural process that already safely sequesters carbon in a form that could remove it from the atmosphere for thousands of years.

"At this point, time is of the essence," said Hochella. "To combat rising temperatures, we must decrease CO ₂ levels on a global scale. Examining all our options, including using the oceans as a CO ₂ sink, gives us the best chance of cooling the planet."

Pulling insights from the literature

In their analysis, the researchers argue that engineered nanoparticles offer several attractive attributes. They could be highly controlled and specifically tuned for different ocean environments. Surface coatings could help the particles attach to plankton. Some particles also have light-absorbing properties, allowing plankton to consume and use more CO ₂. The general approach could also be tuned to meet the needs of specific ocean environments. For example, one region might benefit most from iron-based particles, while silicon-based particles may be most effective elsewhere, they say.

The researchers' analysis of 123 published studies showed that numerous non-toxic metal-oxygen materials could safely enhance plankton growth. The stability, Earth abundance, and ease of creation of these materials make them viable options as plankton fertilizers, they argue.

The team also analyzed the cost of creating and distributing different particles. While the process would be substantially more expensive than adding non-engineered materials, it would also be significantly more effective.

In addition to Hochella, the team included researchers from England, Thailand and several US-based research institutions. The study was supported by the European Research Council under the European Union's Horizon 2020 research and innovation program.

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nature; marine biology; fish; ecology research; global warming; forest; atmosphere; ecology

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Fertilizing the ocean to store carbon dioxide: Iron-based fertilizer, engineered into nanoparticles, could help store excess carbon dioxide in the ocean

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The urgent need to remove excess carbon dioxide from Earth's environment could include enlisting some of our planet's smallest inhabitants, according to an international research team led by Michael Hochella of the Department of Energy's Pacific Northwest National Laboratory. Hochella and his colleagues examined the scientific evidence for seeding the oceans with iron-rich engineered fertilizer particles near ocean plankton.

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Nature-based solutions to fight climate change take a holistic approach that promotes biodiversity and ecosystem preservation. While many efforts have focused on planting trees or restoring wetlands, researchers publishing in Trends in Ecology and Evolution on December 15 advocate for the importance of understanding the carbon sequestration potential of the planet's largest animals -- whales. In their paper, the researchers explore how these marine giants can influence the amount of carbon in our air and waters and potentially contribute to the overall reduction of atmospheric carbon dioxide.

"Understanding the role of whales in the carbon cycle is a dynamic and emerging field that may benefit both marine conservation and climate-change strategies," write the authors, led by Heidi Pearson, a biologist from the University of Alaska Southeast. "This will require interdisciplinary collaboration between marine ecologists, oceanographers, biogeochemists, carbon-cycle modelers, and economists."

Whales can weigh up to 150 tons, live over 100 years, and be the size of large airplanes. Like all living things, their hefty biomass is composed largely of carbon and they make up one of the largest living carbon pools in the pelagic ocean, part of the marine system that is responsible for storing 22% of Earth's total carbon.

"Their size and longevity allow whales to exert strong effects on the carbon cycle by storing carbon more effectively than small animals, ingesting extreme quantities of prey, and producing large volumes of waste products," write the authors. "Considering that baleen whales have some of the longest migrations on the planet, they potentially influence nutrient dynamics and carbon cycling over ocean-basin scales."

Whales consume up to 4% of their massive body weight in krill and photosynthetic plankton every day. For the blue whale, this equates to nearly 8,000 pounds. When they finish digesting their food, their excrement is rich in important nutrients that help these krill and plankton flourish, aiding in increased photosynthesis and carbon storage from the atmosphere.

A blue whale can live up to 90 years. When they die and their bodies fall to the seafloor, the carbon they contain is transferred to the deep sea as they decay. This supplements the biological carbon pump, where nutrients and chemicals are exchanged between the ocean and the atmosphere through complex biogeochemical pathways. Commercial hunting, the largest source of population decline, has decreased whale populations by 81%, with unknown effects on biological carbon pump.

"Whale recovery has the potential for long-term self-sustained enhancement of the ocean carbon sink," the authors write. "The full carbon dioxide reduction role of great whales (and other organisms) will only be realized through robust conservation and management interventions that directly promote population increases."
Story Source:
Materials provided by Cell Press. Note: Content may be edited for style and length.

Journal Reference:

Heidi C. Pearson, Matthew S. Savoca, Daniel P. Costa, Michael W. Lomas, Renato Molina, Andrew J. Pershing, Craig R. Smith, Juan Carlos Villaseñor-Derbez, Stephen R. Wing, Joe Roman. Whales in the carbon cycle: can recovery remove carbon dioxide? Trends in Ecology & Evolution, 2022; DOI: 10.1016/j.tree.2022.10.012

Cite This Page:

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dolphins and whales; nature; marine biology; sea life; global warming; air quality; forest; climate

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Whales could be a valuable carbon sink, say scientists

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Nature-based solutions to fight climate change take a holistic approach that promotes biodiversity and ecosystem preservation. While many efforts have focused on planting trees or restoring wetlands, researchers now also advocate for the importance of understanding the carbon sequestration potential of the planet's largest animals -- whales.

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Deep ocean seaweed forests could be the answer to climate change by sucking in carbon and pumping out credits. It's also in demand as a sustainable wonder crop for biofuel or eating.

Some suggest 48 million square kilometres of ocean is suitable for seaweed aquaculture, or 11 per cent of the total ocean area.

And if nine per cent of the oceans were arranged for seaweed carbon sequestration, the atmosphere could return to pre-industrial conditions within decades.

Carbon sequestration involves the permanent removal of carbon from the atmosphere in a "sink" site but growing seaweed for the sole purpose of carbon offsetting or carbon credits has its critics.

However new global pressure on climate change and investor demand for large projects could head off past concerns about high costs, engineering constraints and hostile growing conditions.

People divided over idea

"This is a moonshot solution," researcher Finn Ross told AAP.

"People either say it's a terrible idea, we're wasting valuable feedstock or low-carbon products, or people are saying the opposite, it's an amazing climate solution, let's do it, let's start sinking seaweed."

He says there is significant industry interest in seaweed as a climate solution and it will need to be funded by carbon markets.

"But rather than rushing into it or writing it off completely, there needs to be more oceanic research."

The PhD student has published the latest findings with the head of Deakin University's Blue Carbon Lab, Professor Peter Macreadie, and Sea Green director Paddy Tarbuck.

One of the reasons the industry is in its infancy is the challenge of dropping seaweed by at least 2000 metres.

The paper warns of large research gaps around how much carbon from seaweed makes it into the deep ocean and the impact on sea bottom organisms.

How to grow seaweed still in concept phase

It is also important to consider competition with phytoplankton for nutrients and ending up making little difference overall.

And it must be possible to track changes in oceanic carbon uptake at points in time during seaweed cultivation.

So far, all proposed ideas have been early-stage commercial ventures with no supporting research, according to the paper published in the academic journal Frontiers of Marine Science.

Concepts include tethered growing platforms in the deep ocean or attached to oil and gas infrastructure or offshore wind farms.

Seaweed could be grown at traditional coastal operations and then dumped offshore.

Or untethered automated platforms in the deep seas could rise and fall with ocean currents and to avoid shipping.

Coastline protected by mangroves

Some blue carbon projects already earn credits in Australia's carbon credit market.

But those eligible to absorb and store - or sink - blue carbon are limited to the deep mud of coastal areas where mangroves, salt marshes and sea grasses can act as a natural carbon sink.

Other blue carbon activities, including establishing seaweed, are not currently eligible for credits, the federal environment department said.

Some 18,000 kilometres of the coastline is protected by mangroves, offering protection from storms and tidal surges for 85,000 homes and 175,000 people.

In 2021, coastal mangroves and seagrass sequestered more than 14 million tonnes of carbon - equivalent to emissions from more than four million cars.

Pacific communities produce seaweed as food

Currently,, seaweed production as a food crop provides Pacific communities with an income and could contribute to sustainable development goals that countries including Australia have signed up to.

Planting vast swathes of ocean seaweed solely for carbon credits could be another option, as governments and industry look at new climate solutions.

"It is tough, because they're politically unpopular ... other blue carbon projects are much more popular," Mr Ross said.

But coastal projects have less scope for scaling up, which is the trade-off decision-makers have to make, he said.

An independent review of Australia's carbon credits is due to report by the end of the year, and how to assess any new methods for earning credits will be part of the government's response.
-AAP

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Deep sea 'moonshot' solution for climate

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Deep ocean seaweed forests could be the answer to climate change by sucking in carbon and pumping out credits.

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Smithsonian's National Museum of Natural History

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How Carbon Affects Nearly Everything on Earth – Including Our Future

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In addition to its crucial role for regulating the climate and weather, and providing critical ecosystem services, the ocean is vital to the world's economy, with more than 90% of trade using sea routes and as a source of jobs for millions of people. The ocean is also the stage for a growing range of new ocean-related economic activities and constant innovations.

Key shaping forces impacting the future ocean economy, OECD STI Ocean Economy Group virtual workshop, 31 May 2022

Webinar on value chains in public marine data: A UK case study, 7 September 2021

Virtual workshop on access to research data from public funding: The case of marine data, 28 June 2021

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In addition to its crucial role for regulating the climate and weather, and providing critical ecosystem services, the ocean is vital to the world's economy, with more than 90% of trade using sea routes and as a source of jobs for millions of people.

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Cocoa pod disease threatens chocolate supplies

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Seydou Sorogo is not sure what to do with his cocoa farm. He lives in a country with an economy that was built on the back of cocoa exports, but on farms around his home town Bouafle, in the centre of Ivory Coast, the trees are sick.

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Introduction

Plant viruses are parasites that multiply and survive in plants. Their genomes are too small to effect their own replication and movement throughout plants alone. They must utilize a combination of virus-encoded genes working complementarily with host genes. Thus, viruses have evolved specific genes whose products interact with the host to replicate the virus, other viral gene products to interact with host to allow accumulation and distribution throughout the host plants, and other gene products to interact with vectors to allow transmission to other plants. Viral genes that are involved in replication tend to be conserved, suggesting that replication within a plant cell is rather generic. Indeed, many viruses are able to replicate in protoplasts from plants in which they are unable to systemically invade. In contrast, viral genes involved in spread within plants tend to be much less conserved. This observation suggests that different viruses use different strategies for invading their hosts. Members of the Closteroviridae, which consists of Closterovirus, Crinivirus, and Ampelovirus genera with mono-, bi-, or tripartite genomes, provide some of the better examples of combinations of conserved and unique genes. They all encode a mixture of conserved signature gene modules along with unique genes with no relationship found in other members of the family. The conserved gene products are involved primarily in replication and virion assembly. In fact, some domains and cis-acting elements involved in replication can be exchanged between different viruses. Additionally, members within a genus possess 1-5 unique genes. These gene products are thought to have evolved to interact exclusively with their specific hosts ( Karasev, 2000; Dolja et al., 2006).

There are several unique features of the Closterviridae. First is that they have morphologically polar virions ( Agranovsky et al., 1995; Febres et al., 1996; Tian et al., 1999), which is unique to this virus group. The second feature is that they encode proteins with similarities to molecular chaperones that are required for assembly ( Peremyslov et al., 1999; Alzhanova et al., 2001) and possibly insect interactions ( Tian et al., 1999). However, the most significant feature is that these viruses have evolved to be transmitted similarly, in a semi-persistent manner, but by at least three different types of insect vectors: aphids, whiteflies, and mealybugs. Based on sequence comparisons, they have two conserved gene modules. The first consists of replicase-associated genes including one or two protease (PRO) domains plus methyltransferase- (MT) and helicase- (HEL) like domains and an RNA-dependent RNA polymerase (POL) domain, with the latter being translated by a +1 frame-shift. Although the order of these domains and the large intragenic regions are characteristic of this group of viruses, similar domains occur in most RNA viruses. These gene products are produced from the genomic RNA. The 3′ genes are expressed through subgenomic (sg) RNAs. The second signature gene module consists of five or six genes that encode the major coat protein (CP) and a related minor coat protein (CPm) that varies in size and genomic position among the different viruses plus three other proteins: a protein closely related to the ubiquitous HSP70 proteins ( Karasev et al., 1992; Agranovsky et al., 1997), a small (6 kDa) hydrophobic protein proposed as a membrane anchor, and a protein of ~60 kDa. As noted above, these viruses contain 1-5 non-conserved genes with no relationship to each other.

Citrus tristeza virus (CTV) has a 19.3-kb single-stranded positive-sense RNA genome ( Bar-Joseph et al., 1979; Pappu et al., 1994; Karasev et al., 1995). The genomic RNA of CTV is organized into 12 open reading frames (ORFs), which potentially encode at least 19 final proteins ( Karasev, 2000). Ten 3′ genes are expressed through a nested set of 3′ co-terminal sg mRNAs ( Hilf et al., 1995), which consist of the signature ORFs ( Pappu et al., 1994) plus 5 non-conserved genes (Figure 1).

Figure 1. A schematic diagram of the genetic organization of CTV showing ORFs (open boxes) of each gene. PRO, papain-like proteases; MT, methyl transferase-like domain; IDR, large interdomain region; HEL, helicase-like domain; RdRp, RNA-dependent RNA polymerase domain; HSP70h, analog to heat shock protein; CPm and CP, minor and major coat proteins. Below are expected protein products and the 10 subgenomic RNAs (the ORF translated is shown in parentheses). Left and Right are Northern hybridization analyses of total RNAs hybridized using a 5′-specific probe (left) or a 3′-specific probe (right).

Requirements for Replication

The ten 3′ genes are not required for replication of the genomic RNA ( Satyanarayana et al., 1999). A replicon with only ORFs 1a and 1b plus the 5′ and 3′ non-translated regions (NTR) replicates well in protoplasts ( Satyanarayana et al., 1999). The 5′ NTR is 107 nts and contains a precise structure with two stem loops. This was first noticed when López et al. (1998) analyzed the 5′ sequences of nine different CTV isolates that varied as much as 58%, yet all folded into the same structure. Gowda et al. (2003) found that the precise stem-loop secondary structures, in contrast to the primary sequence, are necessary for replication. In contrast to most other RNA plant viruses, the 3′ NTR does not contain a poly-A tract nor does it appear to fold as a tRNA mimic. Instead it is highly conserved among different CTV strains and is predicted to consist of 10 stem-loop structures with the replication signals within the 3′ 234 nts ( Satyanarayana et al., 2002a). One of the 3′ genes, p23, although not essential, greatly affects the plus-strand to minus-strand ratio of CTV RNAs ( Satyanarayana et al., 2002b). Mutants without a functional p23 gene produce almost equal amounts of negative and positive strands. The wild-type virus produces plus-stranded genomic and sgRNAs ~10-50 times more than minus strands. The absence of a functional p23 gene also reduces or prevents protein production from 3′ genes apparently by preventing the production of single-stranded RNAs to serve as messenger RNAs.

Requirements for Assembly

Although CTV virions had been semi-purified and characterized, only much later was it found that virions consisted of two coat proteins ( Bar-Joseph et al., 1979; Agranovsky et al., 1995; Febres et al., 1996). Most of the virion is encapsidated by coat CP, but ~3% of the virion from the 5′ end is encapsidated by the minor coat CPm ( Satyanarayana et al., 2004). Besides CP and CPm, the HSP70 homolog (p65) and p61 are involved in assembly of virions ( Satyanarayana et al., 2000). Assembly of CPm is initiated at the stem-loop structures in the 5′ NTR and in the presence of HSP70h and p61 encapsidation stops at approximately nt 630 ( Gowda et al., 2003; Satyanarayana et al., 2004). In the absence of HSP70h and p61, encapsidation occurs much more slowly and continues toward the 3′ terminus ( Satyanarayana et al., 2004). Neither protein is active alone. Thus, these two proteins in combination enhance encapsidation by CPm and limit it to the 5′ end of the genomic RNA ( Satyanarayana et al., 2004). Additionally, encapsidation by CPm in the absence of other assembly related proteins shows remarkably high specificity ( Tatineni et al., 2010). Heterologous CPm's with 95-96% amino acid identity from related strains substituted into a CTV replicon with CPm as the only assembly related ORF, generally failed to initiate encapsidation. However, the heterologous CPm in combination with both HSP70h and p61 proteins, but not HSP70h or p61alone, encapsidated at wild-type levels, suggesting that non-specific interaction of CPm and its origin of assembly was mitigated by the combination of HSP70h and p61. Thus, in addition to enhanced virion formation and restriction of CPm encapsidation to the 5′ 630 nts of the genomic RNA, the HSP70h and p61 proteins facilitate encapsidation by heterologous CPm's.

Movement in Citrus Hosts

To establish a productive infection in a host a plant virus needs to be able to move throughout a plant from an initially infected cell. Success depends upon compatible interactions between viral and host factors. Generally, systemic movement is thought to involve two distinct processes: cell-to-cell movement, which is a process that allows the virus to transverse the cell wall between adjacent cells, and long-distance movement, which is a process that allows the virus to enter the sieve element from an adjacent nucleated cell and rapidly move through the connected sieve elements, followed by its exit into another adjacent phloem-associated cell at a distal region of the plant. A major obstacle for the spreading virus is to cross the boundaries represented by the cell wall. For this purpose most viruses utilize specific virus-encoded movement proteins as well as some host proteins that facilitate their translocation through plasmodesmata channels. The viral proteins and their interactions with the host during cell-to-cell movement are fairly well-known (reviewed in Waigmann et al., 2004; Scholthof, 2005; Lucas, 2006). However, the mechanisms of long-distance transport and factors that aid virus entrance into phloem tissue, further vascular movement, and unloading from phloem are much less understood.

CTV generally follows the patterns described above, but the degrees of both cell-to-cell and long-distance movement are more limited than in most well-described systems, and this limitation varies depending on the citrus host. Since CTV infections are limited to phloem-associated cells, the infection can be most easily viewed by looking at fluorescence from green fluorescent protein (GFP)-tagged CTV in peeled bark that exposes phloem cells. In all citrus hosts, long-distance movement appears to be limited to relatively few initial infection sites. In the more susceptible hosts, C. macrophylla and Mexican lime, we estimated that only about 10-20% of the phloem-associated cells were infected ( Folimonova et al., 2008). The number of fluorescent cells in grapefruit and sour orange bark patches was much less, with sweet orange being intermediate. Also, there was a difference in the size of the fluorescent areas. In the more susceptible species, C. macrophylla and Mexican lime, infection sites consisted of clusters of 3-12 cells. In the less susceptible species, sour orange, there were fewer infection sites and they usually were single cells (Figure 2). Sweet orange again tended to be intermediate between these two extremes. Our interpretation is that systemic invasion of CTV begins when the virus enters sieve elements of the phloem, which transport the virus from some distal position in the direction of sugar movement (source to sink), after which at some point the virus exits into an adjacent cell, usually in stems and leaf veins of a new flush. We assume that the adjacent cell is a companion or phloem parenchyma cell, but this differentiation in citrus phloem is not readily apparent, especially when using confocal microscopy of GFP-labeled virus. We refer to this process as "long-distance" movement. We consider the movement of virus to adjacent cells to fill the clusters of multiple cells as "cell-to-cell" movement. Apparently both long-distance and cell-to-cell movement mechanisms of CTV work differently in different citrus species.

Figure 2. Detection of GFP fluorescence in phloem-associated cells of Citrus macrophylla (C mac) and sour orange (So Orange) under a fluorescence-dissecting microscope (center) or a confocal laser scanning microscope showing single cell infections (top) and multiple cell infections (bottom).

In the more susceptible citrus species, CTV also has limited cell-to-cell movement that produces small clusters of infected cells. However, in less susceptible citrus species, it appears that little or no cell-to-cell movement occurs. The virus is able to exit sieve elements but cannot spread to adjacent cells, resulting in infection of isolated single cells. Thus, CTV provides a new pattern of systemic infection in which the virus appears to function with only the long-distance movement mechanism, yet is able to survive in nature. Such a movement pattern has not been described previously. It is not known whether this pattern is characteristic of other members of the Closterovirdae or other phloem-limited viruses.

Aphid Transmission

CTV generally has been moved long distances into new areas by transport of infected planting (or propagating) materials. Prior to the advent of rapid shipping in the nineteenth century, importation of citrus occurred only as seed, avoiding CTV spread as the virus is not transmissible by seed. However, as navigation improved, citrus was moved as plants or budwood, and so was CTV. Presently, the problem is that since even severe isolates are symptomless in some of their hosts, the virus often is spread by well-meaning individuals moving an infected but non-symptomatic plant or budwood from such a plant into a new area. Afterwards, local spread is by aphids, where transmission is in a semipersistent manner. This combination has effectively spread CTV ( Moreno et al., 2008).

Factors affecting aphid transmission include isolate or strain differences of the virus, the aphid species, plant donor and receptor varieties, the environmental conditions, and the number of aphids involved ( Roistacher and Moreno, 1990). In addition, specific isolates or strains of CTV in mixtures may not be equally distributed throughout the source plant, further reducing the likelihood of successful transmission ( D'Urso et al., 2000). Finally, aphids show a marked preference for some citrus species over others, for example it has been observed in feeding choice experiments that Aphis gossypii preferentially infests mandarins or sweet oranges over lemons ( Roistacher et al., 1984). Similarly, A. gossypii exhibited longer feeding periods on Mexican limes than sweet oranges ( Backus and Bennett, 2009), suggesting that host preference can also affect transmission efficiency ( Roistacher and Bar-Joseph, 1984; Hermoso-de-Mendoza et al., 1988; Cambra et al., 2000).

In addition, the observed movement and distribution of CTV correspond with observations of aphid transmissibility from and to specific citrus species. As mentioned earlier, there is a gradient of infection in citrus species, from frequent clusters of infected cells present in C. macrophylla to a scattered distribution of single cells in grapefruit and sour orange. By extrapolation one may suggest the scattered distribution of CTV in the latter species reduces the probability of virus acquisition by the aphid, and the lower titer reduces the chance of successful infection, which explains reports of grapefruit, sweet lime, sour orange, and lemon being both poor donor and receptor hosts ( Bar-Joseph et al., 1977; Roistacher and Bar-Joseph, 1984; Hermoso-de-Mendoza et al., 1988). These differences in aphid transmission rates may have epidemiological consequences in the field ( Moreno et al., 1988; Gottwald et al., 1996).

Silencing of RNAi

Not only must the virus have the capacity to produce proteins that interact with the host to allow cell-to-cell and long-distance movement, it must also have the ability to escape from the host's surveillance system. Plants have evolved an RNA silencing process, one function of which is to protect them against viruses ( Dunoyer and Voinnet, 2005; Wang and Metzlaff, 2005). Viruses generally produce double-stranded RNA sequences that are subject to degradation resulting in production of small RNAs that, in turn, target the homologous sequences in the viral RNA, thus preventing systemic infection. Sometimes the result is a "recovery" phenotype. In turn, viruses generally encode proteins referred to as silencing suppressors that counteract the RNAi plant defense system to allow a systemic infection to be established and maintained ( Voinnet et al., 1999; Roth et al., 2004; Qu and Morris, 2005). Mutations of viral suppressor genes generally result in reduction or prevention of systemic infection ( Chu et al., 2000; Qu and Morris, 2002).

Citrus species utilize RNAi to reduce CTV titer and slow the progress of systemic infection. Thus, as with other viruses, over the course of its evolutionary history, CTV has acquired or adapted genes that exhibit suppression of silencing, namely p20, p23, and CP ( Lu et al., 2004). The CP and p20 gene products function to suppress intercellular silencing, preventing the spread of the silencing signal, and it is presumed, activation of host defenses, while p20 and p23 suppress intracellular silencing and reduce viral degradation. Transgenic expression of p23 has been reported to increase the number and size of infection foci and thus the CTV titer in sour orange, and to release CTV from strict phloem-limitation in sour and sweet orange plants ( Fagoaga et al., 2011). The p23 and CP genes also have additional roles in the viral replication cycle, respectively, control of negative strand accumulation and encapsidation. Even when the virus establishes a systemic infection, some degree of silencing and degradation of the CTV genome occurs, regardless of host species or viral strain ( Ruiz-Ruiz et al., 2011; Harper, unpublished), which raises an important point to be made that host RNAi cannot completely inhibit or eliminate viral replication or infection, and the three suppressors of silencing cannot completely block the RNAi pathway. From an evolutionary perspective this competition has been likened to an "arms race" ( Obbard et al., 2009), and although one would expect the rapidly evolving virus to overcome host RNAi, stabilizing selection may prevent further adaptation, and complete shutdown of the host RNAi pathway would prevent host-cell regulation, leading to severe symptoms and/or death of the plant.

Some Genes are not Needed for Some Hosts

CTV contains five genes, p33, p18, p13, p20, and p23, in the 3′ half of the genome, which are not related to genes of other members of the Closteroviridae. We examined whether these genes are necessary for systemic infection of citrus trees by deleting single genes one at a time ( Tatineni et al., 2008). The deletion of p20 or p23 prevented systemic infection. Apparently both are needed for counter action against the host RNAi resistance mechanism. Additionally, p23 affects replication of CTV RNA ( Satyanarayana et al., 2002b).

However, we found that deletions within the p33, p18, or p13 ORFs individually resulted in no significant loss of ability of the virus to infect, multiply, and spread throughout our common laboratory hosts, C. macrophylla and Mexican lime ( Tatineni et al., 2008). Furthermore, deletions in the p33, p18, and p13 genes in all possible combinations including deletions in all three genes allowed the virus to systemically invade these plants. GFP-tagged CTV with deletions in the p33 ORF or the p33, p18, and p13 ORFs demonstrated that the movement and distribution of these deletion mutants were similar to those of the wild-type virus.

Because CTV was able to move in these hosts by both cell-to-cell and long-distance movement, it is expected that the virus has other genes that function as a minimal set of movement genes for these hosts. Yet, it was not expected that the virus would retain genes that it did not need. We further examined the roles of these expendable genes (p33, p18, and p13) in a wider range of citrus species and relatives within the CTV host range and found that they are needed for systemic infection of some of the hosts ( Tatineni et al., 2011). However, different genes were required for systemic infection of different hosts. The p33 gene was required for systemic infection of sour orange and lemon trees. It would appear that the p33 is involved in interactions with host proteins of sour orange and lemon for successful long-distance transport of CTV. Either the p33 or the p18 gene was sufficient for systemic infection of grapefruit trees. Deletion of both genes prevented systemic infection, but deletion of either one did not. These results suggest that the p33 and p18 gene products provide similar or redundant functions in grapefruit. Similarly, the p33 or the p13 gene was sufficient for systemic infection of calamondin plants, again suggesting that these two gene products provide similar or redundant functions in this host. This property of either of two different genes providing the same function appears to be a rare property for viruses.

Thus, these three genes are required for systemic infection by CTV of its full host range, but different genes are specific for different hosts ( Tatineni et al., 2011). These findings suggest that CTV acquired multiple non-conserved genes for movement and overcoming host resistance and some of these genes (p33, p18, and p13) were gained to extend its host range further.

Induction of Disease Symptoms by CTV

Although viruses of plants have been focused upon because of the diseases they cause, the ultimate interaction when a virus evolves with a host is likely "no disease" or "limited disease." Yet, as viruses interact with plant hosts, they do sometimes cause disease. When disease occurs in a plant, it is often accidental due to the virus moving to a new host presented to it by agricultural practices. Disease symptoms usually occur on portions of the plant that develop and grow subsequent to viral infection. Rarely do symptoms occur in areas of the plant that are fully developed at the time of infection. Disease often results from interference with differentiation or development. Yet, when diseases do occur, they can cause severe damage to plants, and in agricultural crops diseases cause economic losses, sometimes even preventing some crops from being grown.

Examination of a large number of virus isolates (which can be populations of different strains) on a series of different plants from the host range suggested that CTV has the largest number of distinct phenotypes of any plant viruses ( Garnsey et al., 2005; Hilf et al., 2005; Moreno et al., 2008). The number of phenotypes is amplified by the specificity of the phenotypes in different plants. For example, some isolates cause specific symptoms in grapefruit but not other varieties, some in sweet orange and not other varieties, some in both and some in neither. This level of specificity occurs across the whole host range. Besides these disease symptoms seen in the field, vein clearing, leaf cupping, and temporary yellowing and stunting of young seedlings are phenotypes used in greenhouse diagnosis. Yet, it should be kept in mind that the most frequent phenotype is no symptoms.

However, CTV does cause or threaten to cause serious economic damage to all citrus industries. Depending on the virus isolate and the variety/rootstock combination, CTV can cause any of four distinct syndromes ( Bar-Joseph et al., 1989; Bar-Joseph and Dawson, 2008; Moreno et al., 2008). "Decline" results in death of sweet orange, mandarin, or grapefruit varieties on sour orange rootstocks. During the last century, CTV-induced decline destroyed entire citrus industries worldwide, leading to the substitution of the most desirable sour orange rootstock by other rootstocks that are tolerant to CTV decline, but that are inferior for tree growth and fruit production in saline or alkaline soils, and also more susceptible to root pathogens. In contrast, the "stem pitting" (SP) disease caused by CTV results from aberrant phloem development, resulting in visible pits in the wood. This disease does not cause tree death, but substantially reduces vigor and yield of sweet orange and grapefruit trees resulting in chronic yield reductions and high cumulative economic losses. SP is not specific to any particular rootstock. The third CTV-induced syndrome, "seedling yellows" (SY) is characterized by stunting and leaf chlorosis when small sour orange, grapefruit, or lemon trees become infected ( Fraser, 1952). Other varieties do not develop these symptoms. Sometimes, the stunting and chlorosis is so severe that there is a complete cessation of growth. Remarkably, the fourth CTV syndrome in citrus is a complete lack of symptoms in almost all varieties, even including the decline-sensitive sweet orange/sour orange rootstock combination, even though the virus multiplies to high titers. For instance, most citrus trees in Florida are infected with mild isolates that cause no disease symptoms.

Stem Pitting

Interference with differentiation or development results in numerous phenotypes induced by viruses. Lack of chloroplast development that causes chlorosis is probably the most common virus-induced symptom. The reduced photosynthesis causes reduced growth. SP is a common virus-induced phenotype of perennial woody plants that results from interference with stem growth. In healthy and in normally developed areas of infected trees, the cambium, which is between the phloem and xylem, divides and differentiates in opposite horizontal directions producing new xylem on the inward side and new phloem on the bark side resulting in increased girth of the tree trunk and branches. Stem pits develop in areas where development is disrupted. The surrounding areas grow normally leaving the disrupted areas as indented areas or pits. A range of different viruses distributed throughout the plant virus taxon induce SP in a range of plant species, including numerous Prunus species, apples, vinifera grapevines, citrus, and avocado, usually resulting in a slow decline of growth and poor yields. Although this disease phenotype is common in virus-infected perennial woody plants, there is little understanding of the processes that cause the stem pits.

CTV causes SP diseases that greatly limit production in many citrus industries around the world and areas that do not have isolates that cause this disease spend considerable effort to keep it out ( Bar-Joseph et al., 1989; Moreno et al., 2008). Affected trees with severe SP grow poorly, lack vigor, and yield small, unmarketable fruit. Acid limes are very susceptible, sweet oranges and grapefruit also are susceptible, while mandarins are more tolerant. The disease is not associated with scion/rootstock interactions and pitting can occur on either scion or rootstock or both. Citrus production areas in which severe SP isolates are endemic can be productive only by using mild strain cross protection or by not growing susceptible varieties.

Brlansky et al. (2002) found that the formation of pits by CTV apparently is due to the inhibition of production of new xylem in the localized sites affected. The normally developing surrounding areas continue to grow leaving a depression or pit at the affected area. We examined the association of CTV with the formation of stem pits by tagging GFP to the mutants that induced this symptom ( Tatineni and Dawson, 2012). Since CTV has three non-conserved genes (p33, p18, and p13) that are not required for systemic infection of some species of citrus ( Tatineni et al., 2008), this allowed us to examine the effect of deletions of these genes on symptom phenotypes. In the most susceptible experimental host, Citrus macrophylla, the full-length virus causes only very mild SP symptoms. Surprisingly, we found that certain deletion combinations (p33 and p18 and/or p13) induced greatly increased SP, while other combinations (p13 or p13 plus p18) resulted in reduced SP (Figure 3).

Figure 3. Stems of Citrus macrophylla infected with mutants of CTV with all combinations of deletions of the p33, p18, and p13 genes showing different degrees of stem pitting.

One unexpected result was that in severely pitted areas, GFP fluorescence as a marker of virus replication was observed in regions normally made up of mature xylem or wood ( Tatineni and Dawson, 2012). CTV was found in a group of cells that appeared to be on the woody side of the vascular system. In normally developing trees, most of the cells in this area differentiate into tracheary elements, which essentially consists of dead cells with thick walls connected into vessels for water transport. Interspersed in this area are live ray cells that transport nutrients from the phloem. In the full-length virus-infected trees, the fluorescence of GFP always was limited to the phloem ring outside of the cambium layer. However, increased SP was associated with virus-infected cells in areas not normally infected. Since CTV only multiplies and produces GFP in living cells and free GFP was not found in non-infected adjacent cells ( Folimonov et al., 2007), it would not be expected that the virus could produce GFP in mature xylem cells without virus replication nor could GFP made in other cells accumulate in xylem. However, it should be kept in mind that this is a process that occurs over a period of time and the stem increases in girth as the plant grows in the presence of the viral infection. These results suggest that the process of forming a stem pit is not only the lack of producing new xylem in the affected area resulting in a depression in the wood, but also is affecting development and causing cells within the pitted area to continue living and to be susceptible to CTV invasion and replication.

Previously, it was expected that a specific CTV gene product induced SP, and further this product could be used to identify isolates of virus that would cause this disease. In contrast, removal of sequences induced SP. How could deletions in CTV induce severe SP?

Deletion of the p13 ORF tended to be correlated with reduced SP. Thus, deletion mutants that retained the p13 gene (deletion of p33, p18, or p33 plus p18) tended to have the most SP, which might suggest that the p13 gene product was involved in induction of stem pits. However, the triple deletion mutant, which did not have the p13 gene, induced severe SP, demonstrating that interpretation is not so simple. In contrast, increased SP generally was associated with deletion of the p33 ORF. Mutants with the absence of the p33 ORF (deletion of the p33 plus the p18 ORFs, and the p33 plus the p18 and p13 ORFs) induced severe SP. Thus, mutants retaining the p33 gene (deletions of p13, p18, or p13 plus p18) had the least amounts of SP. These results suggest that the presence of the p33 protein could be correlated with reduced SP (its absence increases it). However, the mutant with the deletion of the p18 ORF (p33 and p13 retained) induced moderate SP. Overall, the production of stem pits or no stem pits appears to be related more to a balance between expression of the p33 and p13 and possibly p18 genes ( Tatineni and Dawson, 2012).

In general, deletions in CTV resulted in a substantial increase in the SP disease of citrus. Yet, there are different phenotypes of SP. Some trees have large stem pits that are readily visible in tree trunks and limbs without removing the bark. Other trees exhibit "cheesy bark" SP, which is a high density of very small pits. There is a continuum of levels in between. Some cause rapid decline of tree growth and yield, while others cause little damage to the tree. Additionally, there is the extreme specificity between virus isolates and different citrus species and varieties. It should be noted that most of the other hosts examined did not form stem pits when infected with these mutants ( Tatineni et al., 2008, 2011; Tatineni and Dawson, 2012). There is no reason to think that all of the different SP phenotypes in different citrus hosts would be caused by the same virus-host interactions.

Seedling Yellows

The SY reaction is specific to only certain citrus hosts of CTV during the seedling stage, such as lemons, sour orange, and grapefruit, indicating that there are specific host factors involved in its expression in addition to isolate-specific viral factors. Mild SY symptoms are characterized by slight yellowing of new leaves and slight reduction in growth. Severe SY results in production of very small new leaves following infection. These leaves can be so chlorotic as to be almost white. The plants generally grow no more. Occasionally plants recover from SY and produce a new flush with normal leaves ( Wallace and Drake, 1972).

In Florida, the decline isolate of CTV, T36, induces SY, whereas the widely distributed mild isolate, T30, does not. To delimit the viral sequences associated with the SY syndrome, we created a number of T36/T30 hybrids by substituting T30 sequences into different regions of the 3′ half of the genome of T36 ( Albiach-Martí et al., 2010). Since T36 induces SY symptoms, the objective was to identify sequences that when substituted by T30 sequences would result in not inducing SY. T36/T30 hybrids were used to inoculate sour orange and grapefruit seedlings. Most of the T30/T36 hybrid constructs continued to induce SY symptoms identical to those of T36; however, two hybrids with T30 substitutions of the 3′-most gene (p23) and the 3′ NTR (nucleotides 18,394-19,296) failed to induce SY. This result suggested that the corresponding region of T36 (p23 to the 3′ end) was the determinant of this phenotype ( Albiach-Martí et al., 2010).

Decline

Historically, decline has been the most devastating disease caused by CTV. It caused the death of almost 100 million trees, largely in the Americas early in the last century ( Bar-Joseph et al., 1989; Moreno et al., 2008). It is a man-made disease based on propagation of sweet orange, grapefruit, and mandarins on the sour orange rootstock. This process was largely due to root rot caused by oomycetes of the genus Phytophthora. When growers learned that sour orange was resistant to this disease, industries were converted to this rootstock. This set up a disaster when CTV was brought into the areas in infected propagation materials. Remarkably, the virus does not cause decline in sour orange trees on their own roots, but causes an incompatibility at the graft union that kills other varieties grafted onto this rootstock. Sometimes death can occur in as short a period as a few days, providing the classic picture of a dead tree full of fruit but with no leaves. Yet, the disease easily can be controlled by using alternative rootstocks. However, there are soils in which all other rootstock choices are less desirable in terms of fruit quality and yield.

Decline has been the major problem caused by CTV in Florida because fortunately severe stem-pitting isolates have been kept out so far. Yet, there are soils in which other all other rootstock choices are deficient compared to the sour orange rootstock. Thus, one of our major projects has been to find a way to allow growers to use the sour orange rootstock. Florida has two predominant strains of CTV, a decline strain (T36) and a mild strain (T30). Remarkably the T30 strain does not induce decline. In comparing the two strains, it appears that T36 contains determinants that induce decline that T30 does not have. In an attempt to identify the decline determinants, we have made hybrids in T36 in which T36 sequences are removed and substituted by T30 sequences, similar to the mapping exercise to identify SY determinants. However, this project has lingered due to our inability to assay for decline in the greenhouse with small trees. Under these conditions, sweet orange on sour orange rootstocks grow normally. Apparently, the small trees replace phloem as fast as the virus causes damage to it. We now have a field test on which we await results.

The potential control strategy is to use cross protection (superinfection exclusion: see Folimonova in this series) to protect trees on the sour orange rootstock. Since T36 and T30 are from different strains, T30 cannot be used to protect trees from T36 ( Folimonova et al., 2009). Yet, a non-decline inducing isolate of the T36 strain could be used to protect against the endemic T36 isolates. But we have never been able to find a non-decline isolate of the T36 strain. However, perhaps such an isolate could be made. If we can identify sequences in T36 that induce decline, it should be possible to substitute those sequences from the T30 strain resulting in a T36 hybrid that does not cause decline. This hybrid could be inoculated to the commercial nursery trees on the sour orange rootstock to protect against decline.

RNAi Induction of Symptoms?

Is the viral counter-attack of the host RNAi system a component of disease induction? It has been shown that ectopic expression of one of the CTV suppressors of RNAi, p23, induces virus-like symptoms ( Ghorbel et al., 2001; Fagoaga et al., 2005; see Flores et al., 2013). In addition to intense vein clearing in leaves, transformed Mexican lime plants develop chlorotic pinpoints in leaves, stem necrosis, and collapse ( Ghorbel et al., 2001), which usually are not symptoms associated with CTV infection. Transgenic sour orange plants expressing p23 also develop vein clearing, leaf deformation, defoliation, and shoot necrosis ( Fagoaga et al., 2005). These transgene-induced symptoms differ from the virus-induced symptoms in sour orange. In transgenic limes, symptom severity parallels the accumulation levels of p23, regardless of the source or sequence of the transgene ( Ghorbel et al., 2001; Fagoaga et al., 2005), whereas the symptom intensity in CTV-infected limes depends on the pathogenicity characteristics of the virus isolate. Yet, this difference in the host response could be related to the fact that, in transgenic plants, p23 is produced constitutively in most cells, whereas, in nature, p23 expression associated with virus infection is limited to phloem tissues.

In non-citrus species is has been shown that ectopic expression of viral suppressors of silencing alters mRNA expression levels and induces symptoms ( Soitamo et al., 2011), therefore it may be speculated that suppression of host RNAi defenses alters that plant's small RNA regulatory pathways, resulting in symptom expression ( Pacheco et al., 2012). It frequently has been observed that virus infections trigger an enrichment of both miRNA and passenger miRNA* ( Bazzini et al., 2011; Du et al., 2011; Hu et al., 2011). Virus infections have also been observed to initiate the expression of novel classes miRNA-like small RNAs (ml-sRNA) produced from the stem-loop precursors of conventional miRNAs ( Hu et al., 2011). Changes in the expression of these small RNAs can lead to up or down regulation of their target mRNA ( Pacheco et al., 2012). In virus-infected plants, changes in miRNA expression have been observed to up or down regulate genes involved in regulation of growth and cell differentiation ( Hu et al., 2011; Pacheco et al., 2012). Changes in the accumulation patterns of sRNAs, including miRNAs, have been reported in CTV-infected citrus plants ( Ruiz-Ruiz et al., 2011). Similarly, in citrus there are significant differences in the expression of miRNAs involved in transcription and hormone responses between healthy and CTV-infected plants, although their link to symptom expression remains unknown (Harper, unpublished). Thus, it appears likely that suppression of the host RNAi processes affects symptom production by CTV in at least some of its hosts, but remains an area of future research.

Concluding Remarks

CTV non-conserved genes apparently evolved to allow systemic infection of its hosts. These are genes involved in cell-to-cell and long-distance movement and in counter surveillance. Some are not needed for all hosts. These non-conserved genes can also be involved in induction of disease symptoms. A specific region was mapped to be involved in the SY syndrome. In contrast, deletion of genes was involved in induction of SP in C. macrophylla, apparently causing gene product ratios that induced abnormalities. In both cases, the symptoms resulted from an alteration of development. Interestingly, both of these disease symptoms are non-continuous. SY symptoms usually are transient. Plants respond only briefly and new growth does not exhibit the symptoms. SP is spatially sporadic. Some infected areas develop abnormally resulting in pits, but most other infected areas continue to develop normally.

Viruses evolve to survive in hosts with which they are presented. This involves acquiring and modifying genes to interact precisely with their hosts. A range of potential host species creates a bewildering array of selective factors; each species will differ to some degree in physiology, gene expression, metabolism, and antiviral defenses, and an isolate at an adaptive peak in one host may be less fit in another. The process of adaption to one host may also create the potential to cause disease in another. In citrus for example, most isolates are mild to asymptomatic in pomelo, mandarin, and citron ( Garnsey et al., 1996), which are the three ancestral Citrus species ( Nicolosi et al., 2000) and likely those in which CTV evolved. These same isolates, however, cause an array of symptoms on commercial citrus species, all of which are hybrids of the three ancestral species.

However, the results described above come from simple systems-a pure culture-a single strain of virus from a cDNA clone. Yet, most virus infections in the field are complex populations of mixtures of different strains and defective RNAs. Little is known concerning how these populations equilibrate and which components of the population interact with the host to elicit or prevent disease symptoms. Do components of the population complement to induce disease symptoms? Do some components counteract other components? The next frontier in plant virology is developing an understanding of populations.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

We especially thank all of the people past and present in our laboratories who made this review possible. Funding has been provided by an endowment from the J.R. and Addie Graves family, the Florida Citrus Research and Development Foundation, and the University of Florida Agricultural Experiment Station.

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Ghorbel, R., López, C., Moreno, P., Navarro, L., Flores, R., and Peña, L. (2001). Transgenic citrus plants expressing the Citrus tristeza virus p23 protein exhibit viral-like symptoms. Mol. Plant Pathol. 2, 27-36.

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Roistacher, C. N., Bar-Josehp, M., and Gumpf, D. J. (1984). Transmission of tristeza and seedling yellows tristeza virus by small populations of Aphis gossypii. Plant Dis. 68, 494-496.

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Citrus tristeza virus (CTV) is a phloem-limited virus whose natural host range is restricted to citrus and related species. Although the virus has killed millions of trees, almost destroying whole industries, and continually limits production in many citrus growing areas, most isolates are mild or symptomless in most of their host range.

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Gene Therapy

Javier Chinen, ¹ Joie Davis, ¹ Suk See De Ravin, ² Beverly N. Hay, ¹ Amy P. Hsu, ¹ Gilda F. Linton, ² Nora Naumann, ² Effie Y. H. Nomicos, ² Christopher Silvin, ¹ Jean Ulrick, ² Narda L. Whiting-Theobald, ² Harry L. Malech, ² and Jennifer M. Puck ¹

Abstract

Retroviral gene therapy can restore immunity to infants with X-linked severe combined immunodeficiency (XSCID) caused by mutations in the IL2RG gene encoding the common gamma chain (γc) of receptors for interleukins 2 (IL-2), −4, −7, −9, −15, and −21. We investigated the safety and efficacy of gene therapy as salvage treatment for older XSCID children with inadequate immune reconstitution despite prior bone marrow transplant from a parent. Subjects received retrovirus-transduced autologous peripherally mobilized CD34⁺ hematopoietic cells. T-cell function significantly improved in the youngest subject (age 10 years), and multilineage retroviral marking occurred in all 3 children.

Introduction

X-linked severe combined immunodeficiency (XSCID) is a primary immune deficiency that results from mutations in the IL2RG gene, which encodes the common gamma chain (γc) that is a component of and provides important signaling function for the receptors of interleukins 2 (IL-2), −4, −7, −9, −15, and −21.1-4 Infants with XSCID, who lack T and natural killer (NK) cells and have nonfunctional B cells, die of infections unless treated to establish functional immunity.1 Infants receiving a bone marrow transplant (BMT) from an HLA-matched sibling have almost 95% 5-year survival and achieve significant immune reconstitution.5-7 However, most SCID patients lack an HLA-matched sibling. Although adequate reconstitution can follow HLA-haploidentical parental BM transplantation when performed within 3.5 months of birth, before severe infections occur,8 those receiving parental or unrelated donor BMT later in infancy have 5-year survival rates of only 55% to 75%.4-7,9,10 Furthermore, more than 50% of patients with XSCID or the JAK3-deficient type of SCID treated with BM transplantation fail to develop specific antibody responses, presumably because the presence of nonfunctional autologous B cells in these types of SCID may impede engraftment of functional donor B cells.9,11 Thus, after BM transplantation many XSCID patients remain dependent upon immunoglobulin infusions. Some BMT centers perform BM transplantation for SCID without conditioning, whereas others employ cytoreductive chemotherapy in an attempt to establish donor B-cell engraftment. Chemotherapy may adversely affect patient survival yet may not result in enhanced B-cell engraftment.9-11 Furthermore, late-onset toxicities of chemotherapy include hypodontia, growth delay, and hormonal disturbances.12-14 A subset of patients suffer progressive partial or total loss of their haploidentical T-cell graft over variable periods of time and present with susceptibility to infection, chronic diarrhea and pulmonary diseases, eczema, warts, and growth delay. These problems prompted our exploration of new treatments for these older children with XSCID who have waning immunity after BM transplantation. Gene therapy restored immune function to 12 of 14 infants with XSCID in 2 European trials.1-3 Unfortunately, T-cell leukemias secondary to retroviral vector insertional mutagenesis occurred in 3 patients in 1 of these studies. At least 2 leukemias were in part caused by vector insertional activation of the LMO2 transcription factor gene.2,15,16

While retrovirally-induced leukemia is a serious risk, it must be balanced with the lack of satisfactory alternative treatments for the subset of XSCID children with profound impairment of T-cell immunity because of loss of donor T-cell number and function following haploidentical BM transplantation. These boys suffer progressive, chronic medical conditions despite intensive medical management. We enrolled 3 such older preadolescent XSCID patients in a clinical trial to assess safety and efficacy of treatment with ex vivo retroviral-transduced autologous GCSF-mobilized peripheral-blood CD34⁺ cells.

Patients, materials, and methods

Patients

Subject P1 presented at 6 months of age with untyped Pseudomonas sp and P. jiroveci pneumonia and widespread, chronic dermatitis infected with methicillin-resistant Staphylococcus aureus. His unusual IL2RG mutation at the poly-A addition site allowed expression of trace amounts of normal γc protein. Over a period of 4 years he received 4 T-cell-depleted, haploidentical parent-derived BMTs with no conditioning; however, he never achieved sustained engraftment. Although receiving intravenous immune gamma globulin (IVIG) and antibiotic, antiviral, and antifungal prophylaxis, he developed growth delay, chronic watery diarrhea, numerous upper- and lower-respiratory infections, eczema, and an esophageal stricture. He also had chronic scarring of his right middle lobe from previous recurrent pneumonias, significant loss of major upper-chest venous structures, and development of collateral venous drainage from long-term indwelling central venous catheters. He attended school but had frequent absences, primarily because of the pneumonias and recurrent diarrhea. At the time of gene therapy, no donor engraftment was detected in any lineage including purified mobilized CD34⁺ cells, using the polymerase chain reaction (PCR) quantitative analysis of informative microsatellite DNA sequences ("Quantification of retroviral vector copy number and chimerism analyses").

Subject P2 developed multiple respiratory infections, oral candidiasis, chronic diarrhea, and failure to thrive by 6 months of age. At 11 months, when XSCID was diagnosed, he had graft-versus-host disease (GVHD) from maternal T cells presumably acquired perinatally that caused a rash and permanent hair loss. He received a maternal T-cell-depleted haploidentical BMT without bone marrow conditioning. After 2 years without a major infection, he developed severe diarrhea and failure to thrive. Because of low T-cell counts and poor proliferative responses, a second maternal T-cell-depleted BM transplantation without conditioning was performed. He experienced no improvement and remained dependent on gastrostomy feedings, IVIG, and antibiotic prophylaxis. Despite this prophylaxis, he suffered from recurrent viral and bacterial pneumonias as well as recurrent sinusitis. He has never attended school. At the time of gene therapy, 51% of circulating T cells were of donor (maternal) origin, but there was no detectable engraftment in any other lineage or in mobilized CD34⁺ cells.

Subject P3 had repeated respiratory infections and oral thrush in early infancy and, at 3 months of age, P jiroveci pneumonia. Laboratory studies revealed decreased T-cell counts and no mitogen responses. He received a T-cell-depleted paternal haploidentical BMT with no bone marrow conditioning at age 6 months. He experienced GVHD with rash, loss of fingernails, and diarrhea for 6 months. He continued to receive IVIG and antibiotic prophylaxis but had sinusitis and 1 bacterial pneumonia requiring hospitalization. At 8 years of age, respiratory infections became frequent and bronchiectasis and growth delay were diagnosed. Immunologic studies showed lymphopenia and poor lymphoproliferative responses to mitogens. However, he appeared to retain adequate lymphocyte proliferation to Candida antigen. He continued to suffer from frequent infections of the bronchiectatic areas of his lungs. At the time of gene therapy, 100% of T cells were of donor origin, but no donor engraftment was found in any other lineage or in mobilized CD34⁺ cells.

Informed consent process

The protocol, consent, and minor assent documents were approved by the National Institute of Allergy and Infectious Diseases (NIAID) institutional review board and designated as National Institutes of Health (NIH) protocol 02-I-0057. The protocol was also approved by the NIH Institutional Biosafety Committee and the NIH Office of Biotechnology Activities' Recombinant DNA Advisory Committee; and the US Food and Drug Administration (FDA) approved treatment under its Investigational New Drug (IND) authority.

Informed consent was obtained in accordance with the Declaration of Helsinki. The informed consent process involved 3 stages. First, the principal investigators and staff discussed the protocol with parents and with the potential child subject, following which, the parents and child were given the consent and protocol to read and discuss with the home physician. Several days to weeks later, all components of the protocol and elements of the informed consent and minor assent documents were discussed in detail with the parents and child patient, and the consent and minor assent documents were signed and witnessed. The third stage involved a detailed interview by a member of the NIH Clinical Center patient care Ethics Office to ascertain the level of understanding. A written confirmation regarding adequate understanding was added to the medical chart by the NIH ethics consultant.

Retroviral vector GALV-MFGS-γc

The open reading frame of human IL2RG cDNA was inserted into the NcoI- BamHI cloning site of the murine leukemia virus-based retroviral vector MFGS.17 To obtain gibbon ape leukemia virus (GALV)-pseudotyped MFGS-γc vector, we transduced PG13-packaging cells with MFGS-γc.17 A high-titer producer cell clone was selected and expanded. Supernatant containing vector produced under good manufacturing practices and subject to all of the FDA regulation-required safety testing was stored in 100-mL bags at −70°C. The biologic titer was sufficient in preclinical studies to reproducibly transduce more than 40% of human CD34⁺ cells in a culture containing 90% supernatant and 10% medium. The estimated multiplicity of infection (MOI) for the clinical transductions was 1 to 2.

Collection and transduction of GCSF-mobilized CD34⁺ peripheral-blood stem cells (PBSCs)

Patients received 10 μg/kg of granulocyte colony-stimulating factor (G-CSF; Filgrastim; Amgen, Thousand Oaks, CA) daily for 5 days. Increases in peripheral CD34⁺ cell counts were comparable to those of healthy controls and subjects with chronic granulomatous disease.18,19 Peripheral-blood mononuclear cells (PBMCs) were collected by apheresis on days 4 and 5 of the G-CSF schedule, and CD34⁺ cells were isolated as reported previously.18

CD34⁺ cells were thawed and cultured in flexible and air-permeable bags in serum-free medium (X-VIVO 10; BioWhittaker X-VIVO Media Systems, Cambrex BioScience, Walkersville, MD) supplemented with 1% human serum albumin (GIBCO culture products; Invitrogen, Carlsbad, CA) and 50 ng/mL FLT3 ligand, 50 ng/mL stem cell factor, 25 ng/mL IL-6, 50 ng/mL thrombopoietin, and 5 ng/mL IL-3 (all cytokines purchased from R&D Systems, Minneapolis, MD). Bags had been previously coated with recombinant fibronectin fragment CH296 (RetroNectin, kindly provided by Takara Bio, Shiga, Otsu, Japan). Beginning 16 hours after culture initiation, CD34⁺ cells were transduced 6 hours/day on 4 consecutive days using 100 mL of vector supernatant and 10 mL of medium per 100 × 10 ⁶ cells, supplemented with 6 μg/mL protamine and cytokines at the same concentration as the culture medium. Transduced cells were washed with phosphate-buffer saline (PBS) and resuspended in 20 mL of PBS with 1% HSA.

Quantification of retroviral vector copy number and chimerism analysis

The presence of the retroviral vector in leukocyte lineages was quantified using real-time PCR. Cells were sorted using a Ficoll gradient and immunomagnetic beads (Dynalbeads; Invitrogen, Brown Deer, WI), with specificity for CD3 (T cells), CD19 (B cells), and CD14 (monocytes). CD15-specific beads were used to isolate granulocytes from the red blood cell fraction of the gradient. Genomic DNA was obtained from the sorted cells with QiaAmp DNA blood mini kit (Qiagen, Valencia, CA). Real-time quantitative PCR was performed using primers and labeled probes listed below to quantify the amplification of a specific sequence of the MFGS vector: forward primer, 5′-CGCAACCCTGGGAGACGTCC-3′; reverse primer, 5′-CGTCTCCTACCAGAACCACATATCC-3′; probe, 5′-6-FAM-CCGTTTTTGTGGCCCGACCTGA-TAMRA.

The retroviral copy number was established using a standard curve of increasing numbers of a single clone of transduced K562 cells containing one proviral copy of the MFGS vector per cell. Donor and recipient cells were detected by quantitative analysis of informative microsatellite DNA sequences, as previously described.19

Immunologic evaluation

Lymphocyte proliferation to mitogens and antigens was measured using a carboxy-fluorescein diacetate succinimidyl ester (CFSE) assay.20 T-cell receptor excision circles (TRECs) from total PBMCs were quantified by real-time PCR according to Douek et al.21 We also analyzed the diversity of the T-cell receptor (TCR) Vβ repertoire (spectratyping).22

Safety tests: replication-competent retrovirus and analysis of retroviral insertion sites

PBMC samples were obtained at baseline, every 3 months for the first year, and every 6 months thereafter. Replication-competent retrovirus safety testing was performed on blood cell samples at the Indiana University Vector Production Facility (Indiana University School of Medicine, Indianapolis, IN) where PCR was used to detect retrovirus envelope sequence. In our laboratory, blood cell samples were analyzed to assess the clonality and identity of retroviral insertion sites using 2 methods: linear amplification-mediated PCR (LAM-PCR)23,24 and the linker-mediated PCR (LM-PCR)25 using MseI digestion. Insertion site sequences were aligned to the human genome sequence using the UCSC genome browser ( http://genome.ucsc.edu/).

Results

Patient characteristics before gene therapy

The patients in our study (P1, P2, and P3) had proven IL2RG mutations26; had received from 1 to 4 parental T-cell-depleted BMTs early in life after initial presentation; and had significant impairment of T-cell function. They were treated with gene therapy at 11, 10, and 14 years of age, respectively. Pretreatment histories were notable for growth delay, frequent infections, and chronic diarrhea ( Table 1 ). All patients had very low numbers of CD4⁺ T cells and few or no NK cells; P1 and P2 also had low numbers of CD8⁺ T cells. P1 had no detectable donor engraftment in any blood cell lineage, whereas P2 and P3 had detectable donor engraftment of CD3⁺ T cells only ( Table 1 ). T-cell receptor excision circles (TRECs) that are characteristically found in newly produced T cells21 were undetectable in all 3 patients, and proliferative responses to mitogens were depressed. All 3 patients demonstrated adequate mobilization of CD34⁺ cells from the bone marrow in response to daily administration of granulocyte colony-stimulating factor, allowing hematopoietic stem cells (HSCs) to be harvested from peripheral blood by apheresis. Importantly, the HSC-enriched CD34⁺ cells in all cases were exclusively of patient origin, explaining the absence of durable multilineage engraftment from their previous BMTs (not shown).

Table 1

Patient

P1

P2

P3

Early history

IL2RG mutation

Poly-A addition site

R289X

M145 frame shift

Evaluation prior to gene therapy

CD16/56⁺ CD3 − NK cells/μL, nl 87-505

9

0

3

Transduction of CD34⁺ cells

Initial CD34⁺ cells/kg placed in culture*

2.77×10 ⁷

2.33×10 ⁷

1.79×10 ⁷

Total cells/kg harvested, 80%-90% CD34⁺†

9.5×10 ⁷

8.0×10 ⁷

7.8×10 ⁷

CD34⁺ γc-transduced cells/kg, final product ‡

2.92×10 ⁷

2.85×10 ⁷

3.13×10 ⁷

Ex vivo transduction of patient CD34⁺ PBSCs and infusion of transduced cells

From 17.9 to 27.7 million cryopreserved autologous CD34⁺ cells/kg ( Table 1 ) were thawed as described ("Patients, materials, and methods, Collection and transduction of GCSF-mobilized CD34⁺ peripheral-blood stem cells").27-29 After 4 daily transductions with GALV-enveloped MFGS-γc vector (MOI of 1-2),21 the cells expanded 3.4- to 4.3-fold while remaining 80% to 90% CD34⁺; 39% to 45% of total cells in the culture were MFGS-γc-transduced as assessed by flow cytometry expression of the γc transgene. Based on these measurements, each patient received approximately 30 × 10 ⁶ transduced CD34⁺ cells/kg intravenously. Vector copy number per transduced cell was 1.1 to 3.7.

Gene marking and changes in number and function of immune cell lineages

All 3 patients have retroviral marking in multiple leukocyte lineages ( Table 2 ). Consistent with the selective growth and survival advantage conferred upon T cells by functional γc, there has been consistently higher marking in T cells than in CD15⁺ myeloid cells. At latest measurement, mean provirus copy number per CD3⁺ T cell for P1, P2, and P3, respectively, was 0.06, 1.0, and 0.22. Corrected B cells also exhibit a selective enrichment, although less marked than in T cells ( Table 2 ).

Table 2

Months after gene therapy

P1

P2

P3

T cell

B cell

Myeloid

T cell

B cell

Myeloid

T cell

B cell

Myeloid

3

0.04*

ND

ND

0.17*

ND

ND

0.28

0.10

0

Although T-cell counts have not normalized in any of the patients, P2 has shown improvement in several immunologic parameters ( Table 3 ; Figure 1 ). His CD4⁺ and CD8⁺ T-cell numbers increased ( Figure 1 A); and CD4⁺ CD45RA⁺ naive cells ( Table 3 ) and TRECs from total PBMCs have appeared and persisted ( Figure 1 B), signifying new thymic output. The ratio of host versus donor origin of his T cells also shifted ( Figure 1 B). Before gene therapy, 49% of his very low number of T cells was autologous, whereas 51% derived from his maternal BM transplant. However, at 6 months and thereafter, he had only 13% donor cells. Taking into account his higher total T-cell counts, the maternal donor fraction stayed constant while autologous T cells increased in both absolute and relative number. Normal in vitro proliferation to mitogens PHA, ConA, and PWM and to Candida antigen has also been restored ( Figure 1 D and not shown).20 Finally, following gene therapy, T cells from P2 showed increased T-cell antigen receptor diversity as assessed by Vβ TCR spectratype analysis ( Figure 1 E).22

Table 3

Before treatment

Months after treatment

3

6

9

12

15

18

CD4/CD45RAT cells/μL, nl 31-533

2

4

15

20

14

16

17

CD8/CD45RA T cells/μL, nl 101-636

19

22

37

29

33

54

39

NK cells/μL, nl 87-505

2

17

7

17

29

10

10

No similar improvements in lymphocyte functions were measured in P1 or P3. However, prior to gene therapy, T cells of P3 were 100% of donor origin, but by 9 and 12 months following gene therapy a small number of T cells of host origin became detectable. For all 3 patients, NK cells increased from 0 to 10 cells/μL at baseline to 10 to 20 cells/μL at latest follow-up (not shown). B-cell counts and serum IgA, IgM, and IgE levels did not change, and all 3 patients remain on their pre-gene therapy schedule of periodic intravenous pooled immune globulin treatments.

General clinical status after gene therapy

There have been no complications to date, with 30, 23, and 12 months of follow-up for P1, P2, and P3, respectively. All have reported improved well-being and stamina, reduced frequency of diarrhea and respiratory infections, and resolution of rashes. Furthermore, all have experienced linear growth, P1 gaining 4.9 cm (1.6 cm/year since treatment); P2 gaining 6.9 cm (3 cm/year); and P3 gaining 3.3 cm (3.3 cm/year). P1 and P3 have had fewer school absences than before gene therapy, and home-schooled P2 has had fewer doctor visits. At 6 months after gene therapy, P1 developed 0.5-cm bilateral submandibular lymph nodes and visible tonsillar tissue that have persisted without changing.

Safety monitoring

Safety monitoring has revealed, in addition to negative tests for replication-competent retrovirus, consistently polyclonal assessment of retroviral insertion sites using LAM-PCR; ( Figure 2 )23,24,30 and LM-PCR.25 From a sample of the preinfusion-transduced CD34⁺ cells from P1, 203 insertion sites were isolated, sequenced, and assigned to specific genomic locations. As previously noted for murine leukemia virus vectors, insertions near the 5′ end of genes were more frequent than predicted by random integration models,25,31 but no insertions were found near the LMO2 locus. Similarly, posttreatment peripheral-blood samples from all 3 patients have yielded 260 distinct insertion sites (24 from P1, 219 from P2, and 17 from P3) but none within 5 million base-pairs of LMO2 or ZNF217 and only one 58-kb 5′ to CCND2 (genes suggested to be common integration sites with oncogenic potential).

Discussion

There are no standard treatments for XSCID patients who fail to develop or lose T-cell function after haploidentical BM transplantation. We observed unequivocal immunologic improvement in 1 of our 3 patients treated with gene therapy (P2), and all 3 patients had persistence of transduced T and B cells in peripheral blood for at least 12 to 30 months. To date there have been no adverse effects. Thus, consistent with our preclinical studies in human/sheep chimeras,17 our human XSCID trial has demonstrated that mobilized peripheral CD34⁺ cells can give sustained multilineage marking following ex vivo retroviral transduction and reinfusion. Though myeloid marking waned to less than 1%, reflecting low transduction and/or engraftment rates for HSCs, a significantly higher level of corrected T cells, and to a lesser extent corrected B cells, persisted due to the survival advantage conferred by the γc transgene. Two patients, P2 and P3, maintained T-cell provirus copy numbers of 1.0 and 0.22, respectively, with evidence of new autologous T-cell production; indeed P2 had improvement in several objective measures of T-cell function.

Recently reported gene therapy for 2 XSCID patients at 16 and 20 years of age noted failure to achieve significant T-cell marking.32 This raises the question whether increasing age is a barrier to efficacy of XSCID gene therapy. The protocols for these 2 patients were similar to those successfully used for XSCID infants,1,3 also using bone marrow-derived CD34⁺ cells. Interestingly, neither these published older XSCID patients nor our P1 had experienced a sustained period of normal thymic output of allogeneic T cells after a BM transplantation earlier in life. In contrast to these patients, our P2 and P3 originally had full T-cell reconstitution for at least 2 years following haploidentical BM transplantation. Older XSCID patients who once had but then lost T-cell production in the thymus might be expected to experience better results following gene therapy than those with a thymus that had never been repopulated, including our patient P1. These observations suggest that success of gene therapy in older children after BM transplantation may relate not only to age per se but also to prior successful T-cell production and retained potential functionality of the thymus. Monitoring of thymic output, as indicated by TREC number or CD45RA⁺ naive T-cell counts in post-BM transplantation XSCID patients, might permit an early determination regarding whether a previously successful BM transplantation is failing, so that an intervention such as gene therapy can be performed before further loss of thymic potential. We note that our youngest patient, P2, at 10 years of age experienced the most benefit from gene therapy, and it is possible that intervention with gene therapy even earlier may increase efficacy in post-BM transplantation XSCID patients with waning immunity.

Unlike P2, subjects P1 and P3 have not had improvement in immunologic parameters that can be attributed to gene therapy despite the persistence of marked T cells. Several factors may contribute to their poorer responses. For P1, the low numbers of corrected T cells may be a consequence of the same circumstances that caused his 4 previous BMT rejections. His unusual IL2RG mutation at the poly-A addition signal causes destabilization of mRNA but allows residual trace expression of normal γc protein and development of small numbers of autologous T cells. Prior to gene therapy of P1, his autologous T cells were identified histochemically and by DNA fingerprinting in lymphocytic infiltrates of skin and hair follicles. These sites of rash and alopecia, reminiscent of Omenn syndrome, resolved following gene therapy. P3, treated with gene therapy at 14 years of age, has had persistently higher gene marking of T cells than P1, but lower than seen in P2, consistent with the hypothesis of an effect from age-associated thymic senescence. P3 also had the most restricted pretreatment T-cell repertoire of our 3 patients, consistent with very poor thymic function (spectratyping not shown).

The dose of transduced CD34⁺ cells is likely to affect the level and persistence of transduced T cells, which in turn influences degree of immune reconstitution. The 2 XSCID gene therapy clinical trials in infants suggested that greater than 3 × 10 ⁶ transduced bone marrow CD34⁺ cells/kg were needed for successful immunologic reconstitution.3,15 Moreover, in the published report of unresponsive older XSCID patients, 1 received only 0.7 × 10 ⁶ transduced CD34⁺ cell/kg, whereas the other patient received 4.5 × 10 ⁶ CD34⁺ cells/kg but with a transduction efficiency of only 13%.32 We obtained significantly larger numbers of peripheral-blood CD34⁺ cells from apheresis and thus treated each patient with approximately 30 × 10 ⁶ transduced CD34⁺ cells/kg at approximately 40% transduction efficiency. The higher corrected cell dose may have led to higher and more persistent T-cell marking in all 3 of our patients than in the 2 previously reported older patients.

Though a high dose of transduced cells might confer an increased risk of insertional mutagenesis, it also might be necessary for achieving the level and persistence of marking we have observed. To date we have found no instances of genomic insertion of retrovirus near LMO2 in 463 unique validated junction fragments sequenced from samples obtained before and after infusion of transduced cells. Possible explanations for our different insertion profiles include the following, in addition to ascertainment differences from methods of isolating junction fragments: (i) our MFGS murine leukemia virus vector backbone had 3 engineered stop codons and lacks the mutation in the stem cell-specific repressor binding site16 of the vector in the XSCID gene therapy trial in France; (ii) we used a GALV-pseudotyped vector (as did the XSCID gene therapy trial in England, in which LMO2 insertions and leukemia have also not been reported) that may target a more immature spectrum of CD34⁺ cells than the amphotropic vector used in France3; (iii) a 12-fold lower concentration of IL-3 during transduction culture than the French trial; (iv) older patients rather than infants; and (v) CD34⁺ cells derived from GCSF-mobilized peripheral blood rather than bone marrow. These differences, particularly age, retroviral envelope, and source of CD34⁺ cells, may have resulted in a different composition and/or ratio of transduced early progenitors and true stem cells. To date, the risk factors that allowed leukemias to develop in 1 of the 2 gene therapy trials in XSCID infants are not fully defined. Further follow-up of clinical, immunologic, and molecular parameters in our patients will establish the long-term safety and efficacy of this approach to gene therapy for preadolescents with XSCID who have failed to achieve or maintain immune reconstitution after BM transplantation.

Acknowledgments

This work was supported by the Divisions for Intramural Research, NIAID, and NHGRI, NIH.

We thank our patients and their families; Drs R. H. Buckley, T. Frazier, and H. M. Rosenblatt for referral, advice, and excellent patient care; and K. Hines and other staff of the Department of Transfusion Medicine (DTM), Clinical Center (CC), NIH, for preparation of gene-modified cells. We also thank Takara Bio, Inc (Shiga, Japan) for providing clinical-grade RetroNectin under a Clinical Supply Agreement. This paper is dedicated to the memory of Charlie Carter (DTM, CC, NIH).

Footnotes

An Inside Blood analysis of this article appears at the front of this issue.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734.

Authorship

Contribution: J.C., H.L.M., and J.M.P. designed the study and performed the majority of work in carrying out the clinical trial; J.D., S.S.D.R., B.N.H., and J.U. provided clinical care support; E.Y.H.N. provided regulatory support; S.S.D.R., A.P.H., G.F.L., N.N., C.S., and N.L.W.-T. provided basic laboratory support; and J.C., H.L.M., and J.M.P. wrote the paper.

H.L.M. and J.M.P contributed equally as senior authors to this study.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Harry L. Malech, Laboratory of Host Defenses, NIAID, NIH, Building 10-CRC 5-West Labs, Room 5-3750, 10 Center Drive MSC 1456, Bethesda, MD 20892-1456; e-mail: vog.hin@hcelamh.

References

4. Puck JM. X-linked severe combined immunodeficiency. In: Ochs H, Smith CIE, Puck JM, editors. Primary Immunodeficiency Diseases, a Molecular and Genetic Approach. 2nd ed. New York, NY: Oxford University Press; 2007. pp. 123-136. [ Google Scholar]

9. Buckley RH, Fischer A. Bone marrow transplantation for primary immunodeficiency diseases. In: Ochs H, Smith CIE, Puck JM, editors. Primary Immunodeficiency Diseases, a Molecular and Genetic Approach. 2nd ed. New York, NY: Oxford University Press; 2007. pp. 669-687. [ Google Scholar]

16. Gray JT, Hacein-Bey-Albina S, Cavazzana-Calvo M, Fischer A. The effect of the B2 mutation on hematopoietic reconstitution in the French XSCID gen therapy trial. Mol Ther. 2006;13(suppl 1):S254. [ Google Scholar]

27. Malech HL, Maples PB, Whiting-Theobald N, et al. Prolonged production of NADPH oxidase-corrected granulocytes following gene therapy of chronic granulomatous disease. Proc Natl Acad Sci U S A. 1997;94:12133-12138. [[ PMC free article] [ PubMed] Google Scholar]

30. Schmidt M, Hacein-Bey-Abina H, Wissler M, et al. Clonal evidence for the transduction of CD34[⁺ cells with lymphomyeloid differentiation potential and self-renewal capacity in the SCID-X1 gene therapy trial. Blood. 2005;105:2699-2706. [ PubMed] Google Scholar]

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Gene therapy improves immune function in preadolescents with X-linked severe combined immunodeficiency

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Gene Therapy Javier Chinen, 1 Joie Davis, 1 Suk See De Ravin, 2 Beverly N. Hay, 1 Amy P. Hsu, 1 Gilda F. Linton, 2 Nora Naumann, 2 Effie Y. H. Nomicos, 2 Christopher Silvin, 1 Jean Ulrick, 2 Narda L. Whiting-Theobald, 2 Harry L. Malech, 2 and Jennifer M.

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Origins and evolution of viruses of eukaryotes: The ultimate modularity

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Viruses and other selfish genetic elements are dominant entities in the biosphere, with respect to both physical abundance and genetic diversity. Various selfish elements parasitize on all cellular life forms. The relative abundances of different classes…

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Oops. Something went wrong. Please try again. | Khan Academy

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The past year has seen many a new word popping up in our languages: Furlough (from the Dutch 'verlof' or paid leave), social distancing, lockdown, you name it. Most of these have 'gone viral', just like the virus itself. And just like the virus itself the word 'virus' mutated over time.

The word 'virus' was long known in science, but it was not used to describe the pathogen we know it to be. That was the work of Dutch biologist Martinus Willem Beijerinck.

Beijerinck was the third of three scientists who had worked on the tobacco mosaic disease, an infection that could devastate whole crops. He continued the work done on the disease by Adolf Mayer, former Director of the Agricultural Experiment Station at the Agricultural School in Wageningen where he himself was based. Meyer found that if a bacterium was the cause, there was something strange going on but he could not figure out what it was.

The next step in the right direction was made by Russian botanist Dmitrii Ivanovsky He concluded that the tobacco mosaic disease is caused by something much smaller than a bacterium, because it had slipped through the finest filters of the time, that no bacterium could cross.

He published his findings in several publications, amongst which was an article entitled 'Die Pockenkrankheit der Tabakspflantze', published in Mémoires de l'Académie Impériale des Sciences de Saint-Pétersbourg in 1890.

Apparently this was not picked up by our third man, Beijerinck. He conducted similar research on the tobacco mosaic disease as Ivanovsky had done, but concluded there had to be a new form of infectious agent. Because it was soluble in water Beijerinck called it Contagium vivum fluidum and he called the pathogen 'virus' to distinguish it from bacteria.

He also suggested the new idea that viruses were only capable of reproducing in cells of other organisms. His hypothesis was confirmed a few years later, when electron microscopes became available. I am not sure whether Beijerinck lived to see this new type of kit, because he died in 1931, the year it was invented.

Marja Kingma, Curator Germanic Collections. References and further reading:
Beijerinck, Martinus Willem, Verzamelde geschriften van M. W. Beijerinck ter gelegenheid van zijn 70sten verjaardag ... uitgegeven door zijne vrienden en vereerders. (Delft, 1921-1940.) 6 vols. Shelfmark 12260.l.13.

Iterson Jr. , G. van, Dooren de Jong, L.E. den, Kluyver, A.J., Maritinus Willem Beijerinck. His life and his work. The Hague, 1940. Shelfmark 10761.i.33 Separate publication in English translation of part 2 of vol. 6 of ' Verzamelde geschriften'. Another edition was published in 1983 by Science Tech in Madison, Wisconsin. Shelfmark 85/11941

Iwanowski, D. (1892). "Über die Mosaikkrankheit der Tabakspflanze". Bulletin Scientifique Publié Par l'Académie Impériale des Sciences de Saint-Pétersbourg / Nouvelle Serie III (in German and Russian). 35: pp. 67-70. Translated into English in Johnson, J., Ed. (1942) Phytopathological classics No. 7, pp. 27--30 Neither item held by the BL.

Zaitlin, Milton. The Discovery of the Causal Agent of the Tobacco Mosaic Disease. In: Discoveries in plant biology / S.D. Kung and S.F. Yang (Eds.). Hong Kong, 1998, Chapter 7, pp 105-110. Available at https://www.apsnet.org/edcenter/apsnetfeatures/Documents/1998/ZaitlinDiscoveryCausalAgentTobaccoMosaicVirus.pdf .

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The past year has seen many a new word popping up in our languages: Furlough (from the Dutch 'verlof' or paid leave), social distancing, lockdown, you name it. Most of these have 'gone viral', just like the virus itself. And just like the virus itself the word 'virus' mutated over...

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Some of the worst epidemics and pandemics in history have doomed whole civilizations and brought once powerful nations to their knees, killing millions. While these terrible disease outbreaks still threaten humanity, thanks to the advances in epidemiology we no longer face the same dire consequences as our ancestors once did.

Here are 21 of the worst epidemics and pandemics in history, dating from prehistoric to modern times.
Related: Spanish Flu: The deadliest pandemic in history
1. Prehistoric epidemic: Circa 3000 B.C.

About 5,000 years ago, an epidemic wiped out a prehistoric village in China. The bodies of the dead were piled inside a house that was later burned down. No age group was spared, as the skeletons of juveniles, young adults and middle-age people were found inside the house.

The archaeological site is now called "Hamin Mangha" and is one of the best-preserved prehistoric sites in northeastern China. Archaeological and anthropological study indicates that the epidemic happened quickly enough that there was no time for proper burials, and the site was not inhabited again.

Before the discovery of Hamin Mangha, another prehistoric mass burial that dates to roughly the same time period was found at a site called Miaozigou, in northeastern China. Together, these discoveries suggest that an epidemic ravaged the entire region.

2. Plague of Athens: 430 B.C.

Around 430 B.C., not long after a war between Athens and Sparta began, an epidemic ravaged the people of Athens and lasted for five years. Some estimates put the death toll as high as 100,000 people. The Greek historian Thucydides (460-400 B.C.) wrote that "people in good health were all of a sudden attacked by violent heats in the head, and redness and inflammation in the eyes, the inward parts, such as the throat or tongue, becoming bloody and emitting an unnatural and fetid breath" (translation by Richard Crawley from the book " The History of the Peloponnesian War," London Dent, 1914).

What exactly this epidemic was has long been a source of debate among scientists; a number of have been put forward as possibilities, including typhoid fever and . Many scholars believe that overcrowding caused by the war exacerbated the epidemic. Sparta's army was stronger, forcing the Athenians to take refuge behind a series of fortifications called the "long walls" that protected their city. Despite the epidemic, the war continued on, not ending until 404 B.C., when Athens was forced to capitulate to .

3. Antonine Plague: A.D. 165-180
Related: Plague doctors: Separating medical myths from facts
When soldiers returned to the Roman Empire from campaigning, they brought back more than the spoils of victory. The Antonine Plague, which may have been smallpox, laid waste to the army and may have killed over 5 million people in the Roman empire, wrote April Pudsey, a senior lecturer in Roman History at Manchester Metropolitan University, in a paper published in the book "Disability in Antiquity," Routledge, 2017).

Many historians believe that the epidemic was first brought into the Roman Empire by soldiers returning home after a war against Parthia. The epidemic contributed to the end of the Pax Romana (the Roman Peace), a period from 27 B.C. to A.D. 180, when Rome was at the height of its power. After A.D. 180, instability grew throughout the Roman Empire, as it experienced more civil wars and invasions by "" groups. Christianity became increasingly popular in the time after the plague occurred.

4. Plague of Cyprian: A.D. 250-271

Named after St. Cyprian, a bishop of Carthage (a city in Tunisia) who described the epidemic as signaling the end of the world, the Plague of Cyprian is estimated to have killed 5,000 people a day in Rome alone. In 2014, archaeologists in Luxor found what appears to be a mass burial site of plague victims. Their bodies were covered with a thick layer of lime (historically used as a disinfectant). Archaeologists found three kilns used to manufacture lime and the remains of plague victims burned in a giant bonfire.

Experts aren't sure what disease caused the epidemic. "The bowels, relaxed into a constant flux, discharge the bodily strength [and] a fire originated in the marrow ferments into wounds of the fauces (an area of the mouth)," Cyprian wrote in Latin in a work called "De mortalitate" (translation by Philip Schaff from the book "Fathers of the Third Century: Hippolytus, Cyprian, Caius, Novatian, Appendix," Christian Classics Ethereal Library, 1885).

5. Plague of Justinian: A.D. 541-542

The Byzantine Empire was ravaged by the bubonic plague, which marked the start of its decline. The plague reoccurred periodically afterward. Some estimates suggest that up to 10% of the world's population died.

The plague is named after the Byzantine Emperor Justinian (reigned A.D. 527-565). Under his reign, the Byzantine Empire reached its greatest extent, controlling territory that stretched from the Middle East to Western Europe. Justinian constructed a great cathedral known as Hagia Sophia ("Holy Wisdom") in Constantinople (modern-day Istanbul), the empire's capital. Justinian also got sick with the plague but survived. However, his empire gradually lost territory in the time after the plague struck.

6. The Black Death: 1346-1353

The Black Death traveled from Asia to Europe, leaving devastation in its wake. Some estimates suggest that it wiped out over half of Europe's population. It was caused by a strain of the bacterium Yersinia pestis that is likely extinct today and was spread by fleas on infected rodents. The bodies of victims were buried in mass graves.

The plague changed the course of Europe's history. With so many dead, labor became harder to find, bringing about better pay for workers and the end of Europe's system of serfdom. Studies suggest that surviving workers had better access to meat and higher-quality bread. The lack of cheap labor may also have contributed to technological innovation.

7. Cocoliztli epidemic: 1545-1548

The infection that caused the cocoliztli epidemic was a form of viral hemorrhagic fever that killed 15 million inhabitants of Mexico and Central America. Among a population already weakened by extreme drought, the disease proved to be utterly catastrophic. "Cocoliztli" is the Aztec word for "pest."

A recent study that examined DNA from the skeletons of victims found that they were infected with a subspecies of Salmonella known as S. paratyphi C, which causes enteric fever, a category of fever that includes typhoid. Enteric fever can cause high fever, dehydration and gastrointestinal problems and is still a major health threat today.

8. American Plagues: 16th century

The American Plagues were a cluster of Eurasian diseases brought to the Americas by European explorers. These illnesses, including smallpox, contributed to the collapse of the Inca and Aztec civilizations. Some estimates suggest that 90% of the indigenous population in the Western Hemisphere was killed off.

The diseases helped a Spanish force, led by Hernán Cortés, to conquer the Aztec capital of Tenochtitlán in 1519. Another Spanish force led by Francisco Pizarro conquered the Incas in 1532. The Spanish took over the territories of both empires. In both cases, the Aztec and armies had been ravaged by disease and were unable to withstand the Spanish forces.

When people from Britain, France, Portugal and the Netherlands began exploring, conquering and settling the Western Hemisphere, they were helped by the fact that disease had vastly reduced the size of any indigenous groups that opposed them.

9. Great Plague of London: 1665-1666

The Black Death's last major outbreak in Great Britain caused a mass exodus from London, led by King Charles II. The plague started in April 1665 and spread rapidly through the hot summer months. Fleas from plague-infected rodents were one of the main causes of transmission.

By the time the plague ended, about 100,000 people, including 15% of the population of London, had died. However, this was not the end of that city's suffering. On Sept. 2, 1666, the Great Fire of London started, lasting for four days and burning down a large portion of the city.

10. Great Plague of Marseille: 1720-1723

Historical records say that the Great Plague of Marseille started when a ship called "Grand-Saint-Antoine" docked in Marseille, France, carrying a cargo of goods from the eastern Mediterranean. Although the ship was quarantined, plague still got into the city, likely through fleas on plague-infected rodents.

Plague spread quickly and, over the next three years, as many as 100,000 people may have died in Marseille and surrounding areas. It's estimated that up to 30% of the population of Marseille may have perished.

11. Russian plague: 1770-1772

In plague-ravaged Moscow, the terror of quarantined citizens erupted into violence. Riots spread through the city and culminated in the murder of Archbishop Ambrosius, who was encouraging crowds not to gather for worship.

The empress of Russia, Catherine II (also called Catherine the Great), was so desperate to contain the plague and restore public order that she issued a hasty decree ordering that all factories be moved from Moscow. By the time the plague ended, as many as 100,000 people may have died. Even after the plague ended, Catherine struggled to restore order. In 1773, Yemelyan Pugachev, a man who claimed to be Peter III (Catherine's executed husband), led an insurrection that resulted in the deaths of thousands more.

12. Philadelphia yellow fever epidemic: 1793

When yellow fever seized Philadelphia, the United States' capital at the time, officials wrongly believed that slaves were immune. As a result, abolitionists called for people of African origin to be recruited to nurse the sick.

The disease is carried and transmitted by mosquitoes, which experienced a population boom during the particularly hot and humid summer weather in Philadelphia that year. It wasn't until winter arrived - and the mosquitoes died out - that the epidemic finally stopped. By then, more than 5,000 people had died.

13. Flu pandemic: 1889-1890

In the modern industrial age, new transport links made it easier for influenza viruses to wreak havoc. In just a few months, the disease spanned the globe, killing 1 million people. It took just five weeks for the epidemic to reach peak mortality.

The earliest cases were reported in Russia. The virus spread rapidly throughout St. Petersburg before it quickly made its way throughout Europe and the rest of the world, despite the fact that air travel didn't exist yet.

14. American polio epidemic: 1916

A polio epidemic that started in New York City caused 27,000 cases and 6,000 deaths in the United States. The disease mainly affects children and sometimes leaves survivors with permanent disabilities.

Polio epidemics occurred sporadically in the United States until the Salk vaccine was developed in 1954. As the vaccine became widely available, cases in the United States declined. The last polio case in the United States was reported in 1979. Worldwide vaccination efforts have greatly reduced the disease, although it is not yet completely eradicated.

15. Spanish Flu: 1918-1920

An estimated 500 million people from the South Seas to the North Pole fell victim to Spanish Flu. One-fifth of those died, with some indigenous communities pushed to the brink of extinction. The flu's spread and lethality was enhanced by the cramped conditions of soldiers and poor wartime nutrition that many people were experiencing during World War I.

Despite the name Spanish Flu, the disease likely did not start in Spain. Spain was a neutral nation during the war and did not enforce strict censorship of its press, which could therefore freely publish early accounts of the illness. As a result, people falsely believed the illness was specific to Spain, and the name Spanish Flu stuck.

16. Asian Flu: 1957-1958

The Asian Flu pandemic was another global showing for influenza. With its roots in China, the disease claimed more than 1 million lives. The virus that caused the pandemic was a blend of avian flu viruses.

The Centers for Disease Control and Prevention notes that the disease spread rapidly and was reported in Singapore in February 1957, Hong Kong in April 1957, and the coastal cities of the United States in the summer of 1957. The total death toll was more than 1.1 million worldwide, with 116,000 deaths occurring in the United States.

17. AIDS pandemic and epidemic: 1981-present day

AIDS has claimed an estimated 35 million lives since it was first identified. HIV, which is the virus that causes AIDS, likely developed from a chimpanzee virus that transferred to humans in West Africa in the 1920s. The virus made its way around the world, and AIDS was a pandemic by the late 20th century. Now, about 64% of the estimated 40 million living with human immunodeficiency virus (HIV) live in sub-Saharan Africa.

For decades, the disease had no known cure, but medication developed in the 1990s now allows people with the disease to experience a normal life span with regular treatment. Even more encouraging, two people have been cured of HIV as of early 2020.

18. H1N1 Swine Flu pandemic: 2009-2010

The 2009 swine flu pandemic was caused by a new strain of H1N1 that originated in Mexico in the spring of 2009 before spreading to the rest of the world. In one year, the virus infected as many as 1.4 billion people across the globe and killed between 151,700 and 575,400 people, according to the.
Related: How does the COVID-19 pandemic compare to the last pandemic?
The 2009 flu pandemic primarily affected children and young adults, and 80% of the deaths were in people younger than 65, the CDC reported. That was unusual, considering that most strains of flu viruses, including those that cause seasonal flu, cause the highest percentage of deaths in people ages 65 and older.

But in the case of the swine flu, older people seemed to have already built up enough immunity to the group of viruses that H1N1 belongs to, so weren't affected as much. A vaccine for the H1N1 virus that caused the swine flu is now included in the annual flu vaccine.

19. West African Ebola epidemic: 2014-2016

Ebola ravaged West Africa between 2014 and 2016, with 28,600 reported cases and 11,325 deaths. The first case to be reported was in Guinea in December 2013, then the disease quickly spread to Liberia and Sierra Leone. The bulk of the cases and deaths occurred in those three countries. A smaller number of cases occurred in Nigeria, Mali, Senegal, the United States and Europe, the Centers for Disease Control and Prevention reported.

There is no cure for Ebola, although efforts at finding a vaccine are ongoing. The first known cases of Ebola occurred in Sudan and the Democratic Republic of Congo in 1976, and the virus may have originated in bats.

20. Zika Virus epidemic: 2015-present day

(Image credit: Yuichiro Chino via Getty Images)

The impact of the recent Zika epidemic in South America and Central America won't be known for several years. In the meantime, scientists face a race against time to bring the virus under control. The Zika virus is usually spread through mosquitoes of the Aedes genus, although it can also be sexually transmitted in humans.

While is usually not harmful to adults or children, it can attack infants who are still in the womb and cause birth defects. The type of mosquitoes that carry Zika flourish best in warm, humid climates, making South America, Central America and parts of the southern United States prime areas for the virus to flourish.

21. COVID-19 pandemic: 2019-present day

The ongoing COVID-19 pandemic, driven by the novel coronavirus SARS-CoV-2, may be the world's deadliest viral outbreak in more than a century. From the virus' initial detection in December 2019 to mid-December 2020, the pathogen infected at least 75 million people and caused 1.6 million deaths, Live Science previously reported. As of Sept. 2021, COVID-19 had killed more people in the U.S. than the so-called Spanish flu did during the 1918 flu pandemic.

That said, in total, the 1918 pandemic claimed more than 50 million lives worldwide, out of a global population of roughly 1.8 billion people; the death toll was high, in part, because no vaccines were available at the time and doctors lacked antibiotics to treat secondary bacterial infections. By comparison, today's global population is nearly 8 billion, and as of mid-August 2022, about 6.4 million people had died of COVID-19, although the reported number of confirmed deaths is likely lower than the true total.

To see the updated global case count and number of confirmed deaths from COVID-19, visit the WHO Coronavirus (COVID-19) Dashboard.

Additional resources

Learn what a pandemic is with the Centers for Disease Control and Prevention (CDC). Discover what the coronavirus outbreak can teach us about bringing samples back from Mars. And Learn about how the spread of COVID-19 is fueled through stealth transmission.

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The worst epidemics and pandemics in history

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Discover the deadliest epidemics and pandemics in history - including ones that have wiped out entire civilizations.

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Conserve and sustainably use the oceans, seas and marine resources for sustainable development.

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Ensure healthy lives and promote well-being for all at all ages.

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End hunger, achieve food security and improved nutrition. and promote sustainable agriculture