# data[].attributes.body
Although Gene therapy is only being tested on animals, scientists are hoping that the United States would approve gene therapy in the next couple of years to use on real patients. Another thing that scientists are hoping to accomplish in he near future is improving vectors, which help move the genes to the desired location.

# data[].attributes.attachment_link.readable_text
Ironically, the gene mutations responsible for the suffering of patients with blinding diseases such as retinitis pigmentosa and Leber congenital amaurosis may also hold the key to their treatment. This is the idea behind gene therapy, a cutting-edge approach to treatment that uses genes to either replace genes that are missing in a patient's eye or to produce therapeutic proteins over an extended period of time. In this article, researchers on the forefront of gene therapy provide a glimpse of what gene therapy might look like five years from now, and what hurdles will need to be cleared before we can realize that vision.

Currently, gene therapy operates by injecting the eye with a virus, called a vector, that carries either a healthy gene that will replace the gene that's missing in a certain eye disease, or which enters retinal cells and turns them into "biofactories" that produce a protein that helps treat a disease. Though these approaches are being tried in different ailments, experts say that there are certain diseases that are likely candidates to have the first approved gene therapies in the United States in the next few years.

"I think the one that's most likely to be approved is gene augmentation therapy for the RPE65 form of Leber congenital amaurosis, or for other conditions caused by RPE65 deficiency," says Jean Bennett, MD, PhD, the F.M. Kirby professor of ophthalmology at the Perelman School of Medicine at the University of Pennsylvania, and one of the first researchers to use viral vector-based gene therapy to reverse blindness. She currently is the scientific director of the Phase III human trial of gene therapy for LCA. "I think others will follow, though it's hard to predict the one that will be next because they're all still in Phase I trials. However, I am excited about the data for the treatment of choroidal neovascular complications of age-related macular degeneration using sFlt-1, the receptor for VEGF. And I'm very interested in the studies that are ongoing for Leber hereditary optic neuropathy, and optogenetic therapy. Perhaps therapy for choroideremia would be the second one approved, since that research seems to be moving quickly now." Spark Therapeutics (Philadelphia) is sponsoring the LCA and the choroideremia trials working with the RPE65 gene.

Robert MacLaren, FRCOphth, FRCS, professor of ophthalmology at the University of Oxford in the United Kingdom, says certain types of retinal disease are more amenable to current gene therapy approaches than others, and that might hold true for the next five years. "First, the diseases that are due to deficiencies of genes are probably easier to treat than those in which the genes are causing detrimental effects," he says. "This is because we know a little bit more about gene replacement than gene knockout. Second, a gene that has such a severe effect on the eye that it affects the development of the retina and the normal structure of the eye at birth, such as in Norrie disease, is difficult to treat. This is because the retinas in such diseases can be beyond the point of repair on the day of birth. The only way around that would be to use prenatal genetic screening and begin gene therapy in utero."

A subretinal gene therapy injection requires the creation of a small detachment.

Better Vectors?
Though there are a lot of moving parts to account for in gene therapy, one of the key elements is the vector that's used to transport the gene to the desired location. The vector that's currently used in the overwhelming majority of gene therapy studies is adeno-associated virus-2, though researchers say future vectors might bring different benefits that AAV-2 can't.

To hear it described by Prof. Elizabeth Rakoczy, PhD, director of the molecular ophthalmology department at the University of Western Australia/Lions Eye Institute, the characteristics of AAV-2 make it almost perfectly suited for gene therapy: "At the moment, there are about five different eye diseases being studied in clinical trials by approximately 10 groups, and all of them use a vector that's recombinant AAV-2, a general vector that enters into all the eye's cell types," she says. "The reason for this is that AAV-2 is naturally occurring, is non-toxic in humans-at most causing mild flu-like symptoms-and 80 percent of humans are carriers of it already. Using a virus like this as the basis for your vector is very reassuring, because it has obvious safety benefits." Prof. Rakoczy is also the inventor of Avalanche Biotechnologies' gene therapy agent AVA-101 for wet AMD.

Despite AAV-2's popularity, Prof. Rakoczy says she envisions a future where better vectors emerge. "We need more efficient promoters," she says, referring to the mechanism of the vector that regulates how much of the desired protein is produced in gene therapy. "Many of the present gene therapy approaches fall short of producing sufficient amounts of biologically active proteins. In many cases, we don't see the desired improvement in the patient because even though the protein is produced, it's not in a high enough concentration. That's why the promoter is essential.

"If you really want to look into the future, ideally we'd like to have vectors that are taken up by the target diseased cell with high efficiency and specificity, and switch on automatically when the target protein drops below normal," Prof. Rakoczy adds. "These vectors, in combination with the right promoter, would provide the most proficient biofactory tool set against genetically inherited and complex diseases."

Dr. MacLaren sees vectors as one of the keys to improving gene therapy in the coming couple of years. "There are new variants of the AAV being developed that can be even more efficacious than current ones," he says. "A more efficient virus, for example, means that you can reduce the dose of the virus and still get the same therapeutic effect, and you really need to get those below a certain threshold to deliver the virus without any immune problems or other issues. In the past few years we've actually achieved that goal and now have very efficient viral vectors. We also understand more about the production process, so it's possible to scale up the AAV to good manufacturing process level-or commercial-grade production levels so it can be made for FDA approval. It's all well and good making a virus in your lab and using it for research, but to get it approved for general use in patients you need to have a reproducible GMP that's acceptable to the FDA. We're now able to achieve that."

Dr. Bennett says the development of new viral vectors is one of the areas of gene therapy that's moving quickly. "Researchers are continually modifying the toolkit," she says. "They're adding additional attractive features to the vectors by engineering the viral capsids themselves and by taking advantage of evolutionary approaches to evolve these capsids to have attractive features." The last approach, known as directed evolution, sounds almost like science fiction: Researchers create viruses in a lab in a way that maximizes their diversity, and then promote the evolution of viruses that have the traits that they want through a sort of artificial natural selection. One team of researchers was able to engineer a variety of AAV that delivered its gene cargo in a widespread fashion to the outer retina and rescue disease phenotypes of X-linked retinoschisis and LCA in mice. It also enabled transduction of primate photoreceptors from the vitreous. ¹

Researchers say one area in which vectors will need to improve in the coming years is very basic, yet very important: their size. "Currently, one of the biggest limitations is the size of the gene that can be packaged within an AAV," Dr. Bennett says. "I think it would be great if there were further developments that allowed these capsids to package larger amounts of cargo. For instance, if we could double the size of what could be packaged inside an AAV, that would be wonderful. There are either large genes that you just can't fit into the vectors, or regulatory sequences that we can't use in the vector to limit the expression of specific cell types because those regulatory sequences are too large to fit inside the vector.

"This size constraint limits the treatment in Stargardt's, for example," Dr. Bennett adds. "Stargardt's therapy is being approached using a virus called lentivirus. Lentivirus barely fits that transgene, but doesn't allow the inclusion of the extended regulatory sequence that could potentially provide even better results. And, in some circumstances, lentivirus isn't the best for targeting photoreceptors because it doesn't target them that efficiently."

In one iteration of gene therapy for treating wet age-related macular degeneration, rAAV.sFlt-1 is injected beneath the retina, turning cells into biofactories that produce the antiangiogenic agent sFLT-1 to reestablish the normal balance between angiogenic (vascular endothelial growth factor) and antiangiogenic (sFLT-1) factors in the retina.

Delivery Approaches
In the coming years, surgeons will solidify their route of delivery of the vectors. They now have two approaches to work with-intravitreal and subretinal-each with its own pros and cons.

The main benefit of intravitreal delivery is that it's technically easier to do: The surgeon injects the dosage of vector right into the vitreous. The drawback is that it may not get the vectors to the site the surgeon wants. Subretinal delivery is more challenging, since it involves injecting into the subretinal space and inducing a small detachment, but it gets the vectors right to their site of action. "I'm a little more skeptical of intravitreal delivery than some other people," says Dr. Bennett. "I think it could theoretically be used to treat the fovea, but not much more than that. Maybe diseases that cause distinct foveal problems, such as achromotopsia, could be targeted most efficiently with it. The other challenge with an intravitreal route is that in order to get to the site you need high doses. Those high doses can cause inflammation, which would limit the benefit one might get if one were to use them. That's my main concern: the toxicity vs. efficacy trade-off."

Prof. Rakoczy thinks this inflammation is a key issue. "If I were to look into the future of gene therapy, I believe subretinal delivery would remain the delivery method of choice," she says. "It's been proven that with intravitreal delivery you activate the immune system, so there might be an immune reaction that removes the vector. With subretinal injection, however, you're injecting into an immune-privileged space, and it seems that this fact is much more powerful than we think. It's been shown by Jean Bennett's work that, following a subretinal injection, you can freely inject into the second eye without rejection. In our own work, we've found that when you inject into the subretinal space, patients who already had been exposed to AAV2 and have neutralizing antibodies present in their blood actually responded better to the treatment than the average patient."

Dr. Bennett says, however, that it's possible intravitreal injection might be useful for the so-called generic gene therapy approaches, where the clinician can treat multiple diseases with the same reagent. "Intravitreal delivery might be useful for the delivery of secretable proteins such as neurotrophic factors or antioxidative stress factors to generate a generic strategy for treating retinal diseases," Dr. Bennett says, "Delivering neurotrophic factor would make the cells healthier and keep them alive longer. That would be beneficial in many diseases because many are slowly progressive. If you could slow down cell death and allow those cells to live 10 years longer than they normally would have, that would be very meaningful. The same rationale would extend to using antioxidative stress approaches, because there's a huge amount of oxygen exposure in the outer retina because it's both exposed to light and has one of the highest blood-flow rates in the body. Finally, one of the more recent findings is that some retinal cells may die in these diseases because they're not getting enough metabolic support-in other words, they're not getting fed enough. Researchers are exploring approaches that may provide more nutrients, sugar and energy to these cells to allow them to function better. This could potentially be delivered in a generic approach."
Another generic option, highlighted by Dr. MacLaren, is the field known as optogenetics, or using gene therapy to switch on a gene that makes a cell light-sensitive, no matter what the genetic cause of the blindness. "It has nothing to do with replacing a normal gene," he explains, "but is quite often explored with abnormal genes that are present in other life forms that have a light-sensitive protein. It might have application in AMD to make the macula more light-sensitive."

Prof. Rakoczy thinks there may be improved instrumentation for the injection in five years, as well, assuming a gene therapy approach is approved. "In the lab, we do subretinal injections in the eyes of mice, which are much smaller than the human eye at 2 mm in diameter," she explains. "To do this, we use a stereotactic system, which means it doesn't depend so much on the ability of the person doing the injection." A stereotactic system is a partially automated system that uses computer-generated, three-dimensional coordinates to locate the area in the eye that needs to be injected. The needle's drive mechanism is fixed to the operating table. When the surgeon activates the mechanism, the needle is mechanically driven into the eye using a screw system while monitoring the depth of penetration on the microscope. "Based on this simple approach, I foresee a more automated system such as this being used, since it's less dependent on the manual handling of the needle by the surgeon, though some would not agree that it's better," Prof. Rakoczy says. "Some might argue that, since in humans the injection is performed under local anesthesia, the surgeon doesn't have the ability to correct for movement with a stereotactic system as a surgeon might."

Experts say that, though it doesn't treat disease in the way gene therapy does, genetic testing will evolve in parallel with treatment, since most gene therapy is targeted at a specific gene mutation or other genetic issue.

"For successful gene therapy in the future, we'll need better genetic testing so that we can test the genes in patients with retinitis pigmentosa or any inherited disease," says Dr. MacLaren. "Then, we'll find mutations and know exactly where they are, because you can't plan a gene therapy program without knowing which gene it is you're going to create. Going back 10 years, we had only diagnosed a fraction of the genes that cause RP in our patients. Now, with next-gen sequencing, we're getting 70 to 80 percent of the genes. So, immediately, the pool of potential patients is orders of magnitude higher than it was in previous years." Dr. MacLaren envisions a future where, as more diseases are diagnosed, physicians can add more mutations to a computerized database so that they're caught even while looking for something else. "For instance, a patient may go to his cardiologist and get gene testing," he says. "The heart doctor might look at the test results and say, 'Hang on, you've got an RP gene here,' and then refer him to us. We can then initiate a treatment possibly 10 years before he would even have been aware of the disease."

Though Prof. Rakoczy notes that there will be challenges with gene therapy, she's sure that it will come to fruition, and it will be a blockbuster. "The change in the pharmaceutical industry is coming," she says. "Within five years, we will see one of the vectors now in trial on the market. The appearance of vector-based 'biofactories' will require a new business model for the pharmaceutical industry. This is because now the drug makers get money from every single pill you put in your mouth. But, when you use gene therapy vectors for treatment, once the biofactory is delivered into the body it takes over treating the patient. Thus, at least in theory, they can only sell the product once for every patient. On the other hand, the cost to develop the system is huge, so even though it will be beneficial to patients and cheaper for the government/health-care system, we don't have a model of how to fund it. Even so, in the future gene therapy will be used more widely for complex diseases such as cancer, heart disease, diabetes and retinal disease. That's what's making patients and governments so excited." REVIEW

Dr. Bennett is the scientific co-founder of and advisor to Spark Therapeutics. Dr. Rakoczy is a consultant to Avalanche. Dr. MacLaren is the academic founder of NightstaRx, a company developing gene therapy treatments.

# data[].attributes.attachment_link.description
Ironically, the gene mutations responsible for the suffering of patients with blinding diseases such as retinitis pigmentosa and Leber congenital amaurosis may also hold the key to their treatment. This is the idea behind gene therapy, a cutting-edge approach to treatment that uses genes to either replace genes that are missing in a patient's eye or to produce therapeutic proteins over an extended period of time.

# data[].attributes.body
Some limitations of gene therapy is the fact that if you could enhance human qualities and capabilities, then normal human life would be altered. Another restriction of gene therapy could be that you could affect your whole gene pool.

# data[].attributes.attachment_link.readable_text
Gene therapy is the process of replacing defective genes in a cell with healthy ones. In simple terms, genes form the basis of heredity. They come as triplets of nitrogenous basis which form amino acids. Amino acids are a composition of proteins which play a crucial role in the functioning of our bodies. Therefore defective genes cause malfunctions in metabolic pathways which become diseases, of which the causes are deeply rooted in our genetic design. Currently there isn't a cure for genetic disorders and treatment is only indicative. This is why there's so much turmoil in the medical arena about the possible effects of gene therapy.

Gene Therapy Treatment

Gene therapy can be categorized in to two different types-somatic cell therapy and reproductive cell also referred to as germline therapy. Somatic cells involves targeting somatic cells for gene replacement, while reproductive cell therapy involves replacing defective genes in reproductive cells with correct genes. Changes made in the genetic makeup of somatic cells is only corrective for the patient. Alterations are however not inherited by offspring of the treated person.

List of Pros of Gene Therapy

1. Untapped Potential
One notable factor that gives gene therapy the edge is the remarkable therapeutic potential it has.

2. Replace Defective Cells
Humans have always been prone to variety of diseases; as one cure is discovered, another disease emerges. We are constantly attacked by newer, more dangerous and vituperative types of germs and pathogens. Although many of such diseases can be treated or cured medically, there is no cure for genetic disorders unless defective cells are replaced by proper ones which is what gene therapy does.

3. Eradicate Disease
Furthermore, targeting reproductive cells can get rid of such defects for good. People with genetic disorders such as Alzheimer's disease, Huntington's disease and Parkinson's disease are among those who require gene therapy.

List of Cons of Gene Therapy

1. Modify Human Capabilities
One con of gene therapy is that it may be used to enhance and modify human capabilities. If this was feasible, standards for normal human life would be altered for good. Even if certain countries experimented with this form of therapy, they could create unstoppable armies much like in those movies we've seen on the big screen.

2. Damage Gene Pool
If gene therapy was performed to a certain degree, it possibly could permanently change the human gene pool. Another thing would also be-this treatment therapy may possibly be for the rich only, and could make the rich more richer and the poor more poorer, indefinitely.

3. Rise in Disorders
While the most common choice as a vector, using the virus for such a purpose is not guaranteed. There's an exact point in the host genome where the right should be brought in and there are no assurance that the viral enzyme responsible for this step will be able to bring in the right genes at the exact point in the host genome. If there's an error in the process, the results could be bring about severe disorders. In addition, the body may destroy the vector perceiving that it is a foreign body.

# data[].attributes.attachment_link.description
Gene therapy is the process of replacing defective genes in a cell with healthy ones. In simple terms, genes form the basis of heredity. They come as triplets of nitrogenous basis which form amino acids. Amino acids are a composition of proteins which play a crucial role in the functioning of our bodies.

# data[].attributes.headline
Gene Therapy in the Future: Heart Transplantation A.S.

# data[].attributes.subject
Gene Therapy in the Future: Heart Transplantation A.S.

# data[].attributes.body
Patients who are in end stage heart failure only have one option to survive-a heart transplant. Heart transplantation is difficult, considering the side effects of immunosuppressive drugs and possibility of rejection.

# data[].attributes.body
Some controversies that surround Gene Therapy are  that if it released and anybody could do it, will it be available at a high price which makes it easier for the rich to do it. People also think that if everybody used Gene Therapy, it could possibly make society less acceptable of the people are come out as different. If you decide to use Gene Therapy to target eggs and sperm cells, this is germline gene therapy.

# data[].attributes.attachment_link.title
What are the ethical issues surrounding gene therapy?

# data[].attributes.attachment_link.description
The idea of germline gene therapy is controversial. While it could spare future generations in a family from having a particular genetic disorder, it might affect the development of a fetus in unexpected ways or have long-term side effects that are not yet known.

# data[].attributes.body
AAV2 vectors, or Adeno-associated virus, is a viral single strand DNA genome that is popularly used for testing types of gene therapy. This gene is known for specifically integrating into a single gene, which means long term expression in the tissues without associated problems.

# data[].attributes.attachment_link.readable_text
Abstract

Summary: The unique life cycle of adeno-associated virus (AAV) and its ability to infect both nondividing and dividing cells with persistent expression have made it an attractive vector. An additional attractive feature of the wild-type virus is the lack of apparent pathogenicity. Gene transfer studies using AAV have shown significant progress at the level of animal models; clinical trials have been noteworthy with respect to the safety of AAV vectors. No proven efficacy has been observed, although in some instances, there have been promising observations. In this review, topics in AAV biology are supplemented with a section on AAV clinical trials with emphasis on the need for a deeper understanding of AAV biology and the development of efficient AAV vectors. In addition, several novel approaches and recent findings that promise to expand AAV's utility are discussed, especially in the context of combining gene therapy ex vivo with new advances in stem or progenitor cell biology.

INTRODUCTION

Adeno-associated virus (AAV) vectors are currently among the most frequently used viral vectors for gene therapy. At recent meetings of the American Society for Gene Therapy, nearly half of the presentations involved the use of AAV. This represents a significant turnaround. Historically, AAV has not been of great medical interest, because it has not been identified as a pathogen; thus, the lack of widespread knowledge of the virus initially inhibited its broad use as a vector. Twelve human serotypes of AAV (AAV serotype 1 [AAV-1] to AAV-12) and more than 100 serotypes from nonhuman primates have been discovered to date. The lack of pathogenicity of the virus, the persistence of the virus, and the many available serotypes have increased AAV's potential as a delivery vehicle for gene therapy applications. This review will focus on the biology of AAV and its use as a vector for gene therapy.

BIOLOGY OF AAV

Virus Classification

AAV is a small (25-nm), nonenveloped virus that packages a linear single-stranded DNA genome. It belongs to the family Parvoviridae and is placed in the genus Dependovirus, because productive infection by AAV occurs only in the presence of a helper virus, either adenovirus or herpesvirus. In the absence of helper virus, AAV (serotype 2) can set up latency by integrating into chromosome 19q13.4, establishing itself as the only mammalian DNA virus known to be capable of site-specific integration.

AAV-2 Genome

The AAV-2 genome is a linear, single-stranded DNA of 4.7 kb (Fig. ) ( 60). Both sense and antisense strands of AAV DNA are packaged into AAV capsids with equal frequency. The genome is structurally characterized by 145-bp inverted terminal repeats (ITRs) that flank two open reading frames (ORFs) (Fig. ).

The first 125 nucleotides of the ITR constitute a palindrome, which folds upon itself to maximize base pairing and forms a T-shaped hairpin structure. The other 20 bases, called the D sequence, remain unpaired. The ITRs are important cis-active sequences in the biology of AAV. A key role of the ITRs is in AAV DNA replication. In the current model of AAV replication, the ITR is the origin of replication and serves as a primer for second-strand synthesis by DNA polymerase. The double-stranded DNA formed during the synthesis, called replicating-form monomer, is used for a second round of self-priming replication and forms a replicating-form dimer. These double-stranded DNA intermediates (replicating-form monomer and replicating-form dimer) are processed via a strand displacement mechanism, resulting in single-stranded DNA used for packaging and double-stranded DNA used for transcription. Critical to the replication process are the Rep binding elements (RBEs) (RBE and RBE′) and a terminal resolution site (TRS), which is located within the ITR (Fig. ). These features are used by the viral regulatory protein Rep during AAV replication to process the double-stranded intermediates. In addition to their role in AAV replication, the ITR is also essential for AAV genome packaging, transcription, negative regulation under nonpermissive conditions, and site-specific integration.

The left ORF contains the Rep gene, which produces four Rep proteins, Rep78, Rep68, Rep52, and Rep40. The larger Rep proteins (Rep78 and Rep68) are produced from transcripts using the P5 promoter, whereas the smaller Rep proteins (Rep52 and Rep40) are produced from transcripts using the P19 promoter. Rep78 and Rep68 are produced from unspliced and spliced transcripts, respectively, and are important regulatory proteins that act in trans in all phases of the AAV life cycle. Specifically, they positively and negatively regulate AAV gene expression in the presence or absence of helper virus, respectively, and are required for DNA replication ( 48). The smaller Rep proteins, Rep52 and Rep40, produced from unspliced and spliced transcripts, respectively, are involved in the accumulation of single-stranded viral DNA used for packaging within AAV capsids. All four Rep proteins possess helicase and ATPase activity. In addition, the larger Rep proteins possess strand- and site-specific endonuclease activity (nicking at the TRS) and site-specific DNA binding activity (binding at the RBE).

The right ORF contains the Cap gene, which produces three viral capsid proteins (VP1, VP2, and VP3) using the P40 promoter. Alternative splicing of the P40 transcript is used to produce the three viral proteins from two transcripts. The unspliced transcript produces VP1 (87 kDa), the biggest of the capsid proteins. The spliced transcript produces VP2 (72 kDa) and VP3 (62 kDa). VP2 is produced using a nonconventional ACG start codon, whereas VP3 is produced using a downstream conventional AUG codon. The AAV-2 capsid comprises 60 viral capsid proteins arranged into an icosahedral structure with symmetry equivalent to a triangulation number of 1. The capsid proteins (VP1, VP2, and VP3) are present in a 1:1:10 molar ratio.

AAV Life Cycle

There are two stages to the AAV life cycle (Fig. ) after successful infection, a lytic stage and a lysogenic stage. In the presence of helper virus (adenovirus or herpesvirus), the lytic stage ensues. During this period, AAV undergoes productive infection characterized by genome replication, viral gene expression, and virion production. The adenoviral genes that provide helper functions regarding AAV gene expression have been identified and include E1a, E1b, E2a, E4, and VA RNA. Herpesvirus aids in AAV gene expression by providing viral DNA polymerase and helicase as well as the early functions necessary for HSV transcription. Although adenovirus and herpesvirus provide different sets of genes for helper function, they both regulate cellular gene expression, providing a permissive intracellular milieu for AAV productive infection.

In the absence of adenovirus or herpesvirus, there is limited AAV replication, viral gene expression is repressed, and the AAV genome can establish latency by integrating into a 4-kb region on chromosome 19 (q13.4), termed AAVS1 ( 36, 37). The AAVS1 locus is near several muscle-specific genes, TNNT1 and TNNI3 ( 16). The AAVS1 region itself is an upstream part of a recently described gene, MBS85. The exact function of this gene is not clear, but its product has been shown to be involved in actin organization ( 64). Whether AAV integration into this site is suitable for human gene therapy applications remains to be evaluated. Tissue culture experiments suggest that the AAVS1 locus is a safe integration site.

AAV-2 Site-Specific Integration

One of the features of AAV is its ability to specifically integrate to establish latent infection. Current AAV vectors do not have this ability, and the development of such a vector would ensure long-term transgene expression in tissues without problems associated with insertional mutagenesis.

Some of the viral and cellular requirements for targeted integration have been elucidated. The AAV components that are required have been identified. These include the ITRs (in cis), Rep78 or Rep68 (in trans), and a 138-bp sequence termed the integration efficiency element (IEE), located within the P5 promoter in cis ( 49). It is unclear if the entire 138-bp IEE in P5 is required, since a recent study showed that a 16-bp RBE in P5 is sufficient ( 18). Latent infection with wild-type AAV-2 appears to be nonpathogenic in tissue culture, when Rep is expressed under its own promoter. Such expression is regulated by negative feedback. Excess Rep expression has been shown to arrest cell division ( 75) and induce cellular apoptosis ( 58).

A 33-bp minimum AAVS1 sequence, which contains an RBE-like and a TRS-like sequence separated by 8 nucleotides, is necessary and sufficient to target AAV integration ( 26). The intervening sequence may be varied, but a central 5′ CTC is required. The actual integration site is somewhat downstream from the target sequence and can be variable. Many RBEs have been identified in the human genome, with AAVS1 being the only site that has an RBE and a TRS in close proximity to one other. Interestingly, the AAV genome and AAVS1 can be tethered to each other via Rep68 in vitro ( 68). These observations provide a molecular explanation for why AAVS1 is targeted, even though the exact mechanism remains unknown.

The process of site-specific integration is not completely specific even under ideal conditions of Rep78 and Rep68 expression, with approximately 40 to 70% of integrants occurring in AAVS1. Moreover, the mechanism is imprecise, as judged by there not being reproducible breakpoints for vector-AAVS1 junctions; however, clusters of integrants appear within a 2-kb fragment of AAVS1. While the cis- and trans-acting viral factors required for site-specific integration have been identified, much less is known about cellular factors that are required or may be involved. Only recently has a study provided evidence that a cellular protein, human immunodeficiency virus transacting response element-RNA loop binding protein 185 (TRP-185), can promote AAV integration into AAVS1 further downstream from the RBE via interactions with both Rep and AAVS1 ( 73). Moreover, site-specific integration has been demonstrated in mice and rats transgenic for AAVS1, suggesting that the AAVS1 open-chromatin structure is maintained in vivo and that the cellular factors that mediate site-specific integration are present in nondividing cells ( 56).

AAV Infection

AAV-2 gains entry into target cells by using the cellular receptor heparan sulfate proteoglycan ( 62). Internalization is enhanced by interactions with one or more of at least six known coreceptors including α Vβ ₅ integrins ( 63), fibroblast growth factor receptor 1 ( 53), hepatocyte growth factor receptor ( 35), α vβ ₁ integrin ( 7), and laminin receptor ( 3). The cellular events that mediate AAV trafficking postentry are not completely characterized. Cells defective for dynamin significantly hindered AAV-2 infection, suggesting that AAV is endocytosed into clathrin-coated vesicles ( 15). For successful AAV infection, AAV particles need to escape these endocytic vesicles. Infection experiments with bafilomycin A1 (a drug that inhibits the proton pump for endosomes) suggested that the low pH in the endosomes is essential for virus escape and successful infection ( 9). Moreover, cellular signaling involving the activation of the Rac1 protein and the phosphatidylinositol 3-kinase pathway is necessary for intracellular trafficking of AAV particles using microtubules ( 57). Interestingly, a conserved phospholipase A ₂ motif identified in the N terminus of the VP1 protein was reported to be important for successful infection ( 27). Specifically, the phospholipase A ₂ motif seemed to be playing a crucial role during AAV trafficking, possibly helping AAV escape the late endosome. Mutational analysis of the AAV capsid structure indicated that the fivefold pore structure may also serve as the site for phospholipase domain presentation during viral infection ( 10). Moreover, endosomal cysteine proteases, cathepsins B and L, have implied roles in AAV trafficking and capsid disassembly ( 4). Exactly how AAV enters the nucleus after escaping the endosome is not known and is currently an active area of research. Although AAV is theoretically small enough to enter the nucleus via the nuclear pore complex, an early study suggested that AAV entry may be nuclear pore complex independent ( 29).

AAV AS A GENE THERAPY VECTOR

There are several considerations for any viral vector. These include the ability to attach to and enter the target cell, successful transfer to the nucleus, the ability to be expressed in the nucleus for a sustained period of time, and a general lack of toxicity. AAV vectors have been highly successful in fulfilling all of these criteria. Moreover, a variety of modifications have served to enhance their utility. Several considerations have guided the development of current AAV vectors, especially the lack of pathogenicity of the wild-type virus and its persistence.

The small size of the AAV genome and concerns about potential effects of Rep on the expression of cellular genes led to the construction of AAV vectors that do not encode Rep and that lack the cis-active IEE, which is required for frequent site-specific integration. The ITRs are kept because they are the cis signals required for packaging. Thus, current recombinant AAV (rAAV) vectors persist primarily as extrachromosomal elements ( 1, 59).

rAAV vectors for gene therapy have been based mostly on AAV-2. AAV-2-based rAAV vectors can transduce muscle, liver, brain, retina, and lungs, requiring several weeks for optimal expression. The efficiency of rAAV transduction is dependent on the efficiency at each step of AAV infection: binding, entry, viral trafficking, nuclear entry, uncoating, and second-strand synthesis. Inefficient AAV trafficking ( 30) and second-strand synthesis ( 19) have been identified as being rate-limiting factors in AAV gene expression. Interestingly, the binding of cellular protein FKBP52 to the AAV ITR inhibits second-strand synthesis, and this inhibition is dependent on the phosphorylation state of FKBP52 ( 51, 52, 80). Moreover, epidermal growth factor receptor kinase signaling has been implicated in regulating both AAV trafficking and second-strand synthesis ( 80).

Several novel AAV vector technologies have been developed to either increase the genome capacity for AAV or enhance gene expression (Fig. ). The idea of trans-splicing AAV vectors has been used to increase AAV vector capacity ( 74). This system takes advantage of AAV's ability to form head-to-tail concatemers via recombination in the ITRs. In this approach, the transgene cassette is split between two rAAV vectors containing adequately placed splice donor and acceptor sites. Transcription from recombined AAV molecules, followed by the correct splicing of the mRNA transcript, results in a functional gene product. This application becomes useful for using AAV to deliver therapeutic genes up to 9 kb in size. trans splicing has been successfully used for gene expression in the retina ( 55), the lung ( 39), and, more recently, muscle ( 25). trans-Splicing vectors are less efficient than rAAV vectors.

The design and use of self-complementary AAV (scAAV) vectors to bypass the limiting aspects of second-strand synthesis have been described ( 44). The rationale underlying the scAAV vector is to shorten the lag time before transgene expression and potentially to increase the biological efficiency of the vector. scAAV vectors can fold upon themselves, immediately forming transcriptionally competent double-stranded DNA. One consequence of the use of scAAV is that the maximal size of the transgene is reduced by ∼50% (2.4-kb capacity), but up to 3.3 kb of DNA can be encapsidated ( 71). Rapid transduction has been observed using scAAV in both tissue culture and in vivo experiments.

Many clinically relevant tissues are not susceptible to infection by AAV-2. Greater gene expression was seen in muscle, retina, liver, and heart using AAV serotypes 1, 5, 8, and 9, respectively. The cell surface receptors have been identified for only some of the many AAV serotypes: AAV-3 (heparan sulfate proteoglycan), AAV-4 (O-linked sialic acid), and AAV-5 (platelet-derived growth factor receptor). In addition, a 37-kDa/67-kDa laminin receptor has been identified as being a receptor for AAV serotypes 2, 3, 8, and 9 ( 3). The attachment receptors for the other serotypes have not yet been identified. All of these serotypes are potential candidates for testing as vectors for gene therapy. To date, most of the testing has involved serotypes 1 to 9, which have considerable differences at the capsid amino acid sequence level, except for AAV-1 and AAV-6 (Table ), and has succeeded in identifying vectors with widely divergent tissue specificities.

TABLE 1.

The use of the different AAV serotypes in a pseudotyping approach (the genome of one ITR serotype being packaged into a different serotype capsid) has allowed broad tissue tropisms. However, some tissues remain refractory to transduction using available serotypes. This presents a major challenge for AAV-based gene therapy for clinically relevant tissues.

Rational Design of AAV Capsids

A deeper understanding of the AAV capsid properties has made the rational design of AAV vectors that display selective tissue/organ targeting possible, thus broadening the possible applications for AAV as a gene therapy vector. Two approaches have been used for AAV vector retargeting: (i) direct targeting and (ii) indirect targeting. In direct targeting, vector targeting is mediated by small peptides or ligands that have been directly inserted into the viral capsid sequence. This approach has been used successfully to target endothelial cells ( 61, 69). Direct targeting requires extensive knowledge of the capsid structure. Important aspects involve the following: peptides or ligands must be positioned at sites that are exposed to the capsid surface, the insertion must not significantly affect capsid structure and assembly, and it is important that the native tropism be ablated to maximize targeting.

In indirect targeting, vector targeting is mediated by an associating molecule that interacts with both the viral surface and the specific cell surface receptor. The use of bispecific antibodies ( 8) and biotin ( 6, 50) has been described for AAV vectors. The advantages of this approach are that different adaptors can be coupled to the capsid without significant changes in capsid structure, and the native tropism can be easily ablated. One disadvantage of using adaptors for targeting may involve the decreased stability of the capsid-adaptor complex in vivo. The development of efficient AAV targeting vectors will require a better understanding of all aspects of the AAV infection process: binding and entry, viral processing, and nuclear entry and expression. Significant progress has been made in all these categories, and the development of efficient AAV targeting vectors will expand AAV's use as a vector for many clinical applications.

Immune Response to AAV

One of the biggest challenges facing AAV gene delivery is the host immune response. The host defense mechanism at the adaptive level is made up of cell-mediated and humoral immunity. The cell-mediated response functions at the cellular level, eliminating the transduced cells using cytotoxic T cells, whereas the humoral response produces neutralizing antibodies (Nab), preventing the readministration of vector. Almost no innate response is seen in AAV infection ( 76).

Immune response to AAV is primarily a humoral response ( 72). Preexisting Nab in patients, because of prior infection, account for the humoral response seen toward AAV. In a study by Chirmule et al. ( 13), antibodies to AAV were seen in 96% of the subjects (patients with cystic fibrosis [CF] and healthy subjects), and 32% showed neutralizing ability in an in vitro assay. Nab to AAV have been to show limit AAV transduction in liver ( 47) and lung ( 28); however, no such effect was seen in muscle ( 21), brain ( 43), and retina ( 5). Interestingly, the humoral response to AAV may be T-cell dependent; the inhibition of T-cell function using anti-CD4 antibodies prevents Nab formation and allows vector readministration ( 14, 28, 40).

Cell-mediated responses to AAV vectors have been documented, but this response may be dependent on the route of administration ( 11) and AAV serotype ( 67). A potent immune response to AAV-ovalbumin was observed when AAV was administered intraperitoneally, intravenously, or subcutaneously but not when administered intramuscularly. Moreover, AAV-2 has been shown to induce a weak cell-mediated immune response. This may be attributed to AAV inefficiently infecting mature dendritic cells (DC); however, a recent study demonstrated an efficient infection of immature DC and generated a cytotoxic-T-lymphocyte (CTL) response when used in adoptive transfer experiments ( 77). The extent to which mature and immature DC are transduced by AAV in vivo and the mechanism of how AAV induces a cellular immune response are not known.

In a recent clinical trial for hemophilia B, an unexpected liver toxicity was observed and was attributed to a CTL response to AAV-2-transduced hepatocytes ( 42). Subsequently, it was discovered that the AAV-2 capsid heparin binding motif was responsible for T-cell activation ( 65). This correlated well with a study in mice that showed that AAV-2 infection can activate a CTL response, whereas AAV-7 and AAV-8 do not ( 67). Moreover, Wang et al. ( 67) suggested that the cross-presentation of input AAV capsids via major histocompatibility complex class I presentation may be playing a role in the observed activation of cytotoxic T cells; however, this response does not diminish transgene expression via the targeted destruction of transduced hepatocytes, a finding confirmed by another group ( 38). Taken together, these studies suggest that immune responses are a major hurdle and that a deeper understanding of AAV-host interactions in humans is required for the efficient use of AAV as a gene transfer vector.

AAV CLINICAL TRIALS

AAV has become increasingly common as a vector for use in human clinical trials; as of now, 38 protocols have been approved by the Recombinant DNA Advisory Committee and the Food and Drug Administration (FDA). The increased popularity of AAV vectors reflects the appreciation of the long-term transgene expression observed in animal models and the relative lack of immune response and other toxicities in the models. Other factors that have played a role in encouraging the use of AAV vectors include the discovery of new serotypes and the appreciation that matching the tissue specificity of the serotype with the presumptive target tissue can greatly enhance the potential effectiveness of therapy. In general, the goal of gene therapy can be classified into one of two categories, the correction of an intracellular defect or the synthesis of a secreted protein, which is active at an extracellular level. In the latter case, the site of protein synthesis would not seem to be critical as long as it has no deleterious intracellular effects and is successfully secreted into blood. This assumption has been tested in clinical trials in which proteins normally synthesized in the liver are now induced to be produced in skeletal muscle. Whether the assumption is correct is still not certain, in part because different vector target sites may induce different host immune responses ( 41, 42).

Despite the small packaging capacity of AAV vectors, clever investigators have devised ways of engineering transgenes and associated regulatory sequences so that their sizes can be reduced sufficiently to allow packaging into AAV capsids. In general, the expectations with regard to minimal toxicity have been met, although there have been two notable exceptions to this, which will be discussed below. To date, no clinical cures have been effected, although there have been anecdotal data that have kept hopes up. Trials that have been concluded or are in progress are listed in Table . Several of these will be discussed below in some detail to illustrate specific points of interest and concern.

TABLE 2.

Initial targets for gene therapy included monogenic diseases in which the gene product either was altered to become nonfunctional or was missing. First among these was CF, a lethal, autosomal recessive disease in which the CF transmembrane regulator (CFTR) is inactivated by mutation. CFTR is a component of the Cl − channel and the lack of functional CFTR affects the transmembrane electrical potential. This leads to the accumulation of thick secretions in the lung coupled with a loss of the normal respiratory epithelial ciliary activity. The primary difficulty is pulmonary, with an increased incidence of pulmonary infection, especially by Pseudomonas aeruginosa. Additional difficulty occurs with pancreatic secretion, but the loss of the pancreatic enzymes can be treated with supplements. Thirteen protocols have been approved for phase I and phase II clinical trials using an AAV vector ( 2, 22, 23, 46, 66). Delivery of the vector was achieved by bronchoscope or by aerosol into the lung and in several cases by delivery to the maxillary sinus (to make measurement of the transmembrane potential, which is affected in CF, possible). The primary and most important observation in early trials was the lack of measurable toxicity and a very modest immune response evoked by the route of pulmonary delivery. Serum antibody was evoked but did not affect the subsequent administration of the vector. The measurement of efficacy in the lung is pretty much restricted to measures of pulmonary function; any improvement that was noted in this manner was not statistically significant. However, in those patients who had vector instilled into the maxillary sinus, it was possible to make a somewhat more direct measurement. The most notable effect was an increase in levels of interleukin-10, a cytokine that is anti-inflammatory, and a concomitant decrease in levels of interleukin-8, which has the opposite effect. Major challenges with vector delivery to the lung through the airway included rapid, regular shedding of the respiratory epithelium, which means that cells that have taken up the vector are fairly quickly lost and that the uptake of the AAV-2 vector in cell culture was mostly through the basolateral surface, which is not very accessible via the airway. Thus, consideration must be given to alternative routes of delivery and the possibility of vectors with alternative serotypes.

A second monogenic disease that could be amenable to gene therapy is hemophilia. Although this disease can be lethal, it is functionally chronic with current modes of therapy. The two common forms are hemophilia A and hemophilia B. Clotting requires a complex series of enzymatic reactions. Two of the required enzymes are factors VIII and IX; a lack of the former results in hemophilia A, and a lack of the latter results in hemophilia B. Initial efforts concentrated on the replacement of factor IX, because the coding region and regulatory sequences could readily be encapsidated in the AAV vector. A factor IX AAV vector could be used to "cure" mice with hemophilia B ( 31) and, more excitingly, also performed well in a canine model of hemophilia ( 32). Initial phase I studies were performed by the intramuscular injection of an AAV-2 vector ( 36, 41). Disappointingly, although no vector toxicity was noted, no transgenic factor IX could be detected in serum. The notion had been that although factor IX is normally expressed in hepatocytes, the expression of factor IX, which could be excreted, in muscle cells could raise serum factor IX concentration to a therapeutic level (∼10% of normal). The consequence of the failure to see a rise in the factor IX serum level was to alter the vector target to the liver, the normal site of synthesis, with administration via the hepatic artery ( 42). Vector was administered to the patients in increasing amounts. At the first two doses, there was no detectable toxicity nor any detectable transgenic factor IX in the serum. At the highest dose (2 × 10 ¹² vector genomes/kg), there was detectable transgenic factor IX in the serum for 4 to 9 weeks in the two subjects. However, in contrast to what was observed in animal models, the serum concentration went back to baseline levels. More troublesome was a rise in liver transaminases in the serum, a sign of liver inflammation. Subsequently, the inflammatory response was shown to be caused by the induction of a CTL response ( 45). The first question was whether the immune response was due to the transgene product or the vector. It turned out to be due to the AAV capsid. While an antibody response to capsid had been anticipated, the CTL response to AAV proteins had not been anticipated, because all AAV genes had been deleted from the vector. The working hypothesis is that at the highest dose, where the inflammatory response had occurred, the multiplicity of infection (MOI) was sufficiently high that degradation products of the capsid were displayed on the surface of the transduced hepatocytes in sufficient quantity to induce the CTL response. Thus, there is a conundrum: with the vectors used, the dose required to produce a detectable level of factor IX was also sufficient to induce a CTL response, which destroyed the cells expressing factor IX. Possible solutions to this problem include being able to induce tolerance to the AAV capsid fragments displayed on the surface of the hepatocytes or developing a more efficient vector, which would enable a much lower MOI or dose so that a CTL response would not be evoked. The latter may be able to be achieved by use of alternative AAV serotypes such as AAV-8 or by modification of the surface of the AAV capsid to render trafficking of the ingested AAV particle to the cell nucleus, with ensuing expression of the transgene being much more efficient. An example of the latter approach will be described below in the section on future prospects for AAV vectors.

A much more serious problem arose in a clinical trial involving rheumatoid arthritis ( 33, 70). Rheumatoid arthritis is a disabling inflammatory disease in which the immune system reacts against the body's joint tissue. Current therapy involves blocking the host response against itself. One way of achieving this inhibition is to counteract the effects of the cytokine tumor necrosis factor alpha (TNF-α) by use of the drug adalimumab (Humira). Repeated use of the drug is required whenever there is an exacerbation of the disease in a particular joint. An alternative approach would be to design an AAV vector that could express a TNF inhibitor for an extended period of time, with expression located primarily in the joint that had the vector injected. Promising data were achieved in the animal model of disease. Unfortunately, in the phase I clinical trial, one patient became extremely ill the day after the administration of the AAV vector and died within 4 days. Subsequent investigation established that the patient had died of an overwhelming Histoplasmosis capsulatum fungal infection. The patient had also been treated with adalimumab, one of whose side effects is known to be sepsis. Thus, the question was what role, if any, that the AAV vector played in the demise of the patient. While this was studied, the clinical trial was put on hold by the FDA. The study showed that the patient had already had a systemic histoplasmosis infection before the injection of the vector and that this infection was not controlled, most probably because of adalimumab, which is a systemic drug. Among the conclusions of the investigation was that the AAV vector carrying the transgene had not contributed to any toxicity. The possibility remained that the TNF-α inhibitor expressed from the transgene might have contributed to a reduction in the ability of the host immune system to combat the infection; this was deemed to be highly unlikely because little, if any, of the vector was able to be detected outside of the joint that had been injected. Thus, in a relatively short period of time, the FDA essentially exonerated the AAV vector and permitted the clinical trial to resume.

Parkinson's disease, a chronic neurodegenerative disease, has also been an area in which there has been an AAV clinical trial. In Parkinson's disease, a loss of dopaminergic neurons leads to the loss of inhibitory gamma aminobutyric acid-sensitive input to the subthalamic nucleus. Kaplitt et al. ( 34) and Feigin et al. ( 17) described a study in which 12 patients with advanced Parkinson's disease had an AAV vector carrying a transgene encoding glutamic acid decarboxylase injected into the subthalamic nucleus on one side. The therapy was well tolerated, with no adverse effects attributable to gene therapy noted for any of the patients, who had been divided into three groups that received low, moderate, or high doses of the vector. The clinical impression was that motor activity on the treated side was improved significantly relative to the untreated side regardless of dose. No change in cognition was noted. The clinical impression was supported by position emission tomography scan data, which measured the reduced metabolic activity on the treated side, consistent with enhanced inhibition. Of particular interest was that motor improvement was not noted until 3 months postinjection so that it did not seem directly related to trauma associated with the injection. Also very encouraging was that the observed improvement in motor activity persisted for at least 1 year. Although the trial involved an open surgical procedure, the dramatic improvements noted, if consistent and reproducible, suggest that AAV gene therapy for chronic, degenerative neurological diseases has great promise. Additional clinical trials for Parkinson's disease, Alzheimer's disease, and Batten disease have been approved.

From these three examples of the 38 clinical trials that have been approved, two approaches can be noted. On one hand, the original notion of replacing a defective gene in a monogenic disease is exemplified by the trials involving patients with CF or hemophilia B. The second approach is demonstrated in the Parkinson's disease trial, in which the intention was to block the consequences of a chronic disease characterized more by a metabolic defect caused by the lack of dopaminergic neurons rather than a "cure" of the primary lesion.

Two inferences can be drawn from those clinical trials that have already been done. The first is that there has been relatively little toxicity that can be directly attributed to the AAV vector platform. The one area of potential toxicity appears to arise from an inflammatory response involving cytotoxic T cells responding to fragments of the coat proteins from input vector that are presented on the cell surface as major histocompatibility complexes. This has been observed when very high doses of vector were given via the hepatic artery or by intramuscular injection. It is a particularly complex reaction, because dosage, location of injection, and the possibility of induction of tolerance all have to be taken into consideration. Humoral immunity seems to play a role in some instances when the subsequent administration of a vector may be blocked, but toxicity per se has not been a significant observation. Again, the route of administration seems to be important; little humoral immunity has been noted when the pulmonary route is used. The ability of humoral antibody to block vector activity is significant because the seropositivity of the population to AAV is high (∼80 to 90% for AAV-2). However, the discovery of many new AAV serotypes and the ability to package the AAV-2-based vector DNA into many, if not most, of them suggest that preexisting humoral immunity will not pose a significant barrier to therapy.

FUTURE PROSPECTS

Although significant progress has been made in the use of AAV vectors for human gene therapy, several developments are likely to enhance the potential utility of the system. The host immune response remains of concern so that approaches to mitigate the response would constitute a definite advance. One such approach would be to reduce the vector dose required for a therapeutic response. The discovery of additional AAV serotypes is one possibility ( 24). Another is to modify the surface of the vector capsid to include specific ligands for attachment to target tissues (see "Rational Design of AAV Capsids"). Recently, an alternative approach was described by Srivastava et al. ( 79). The particle-to-infectivity ratio of AAV vector preparations usually ranges from 10:1 to 100:1. These ratios reflect, in part, incomplete or empty vector particles. However, an additional reason for the high ratios includes trafficking from the endocytoplasmic vesicle to the nucleus. In the course of trafficking, the vector particle may become ubiquitinated and thus directed to a proteasome for degradation rather than to the nucleus, where the transgene may be expressed. Srivastava's group found that ubiquitinylation and direction to the proteasome require the phosphorylation of tyrosine residues on the surface of the vector capsid. There are seven tyrosines on the surface of the AAV-2 capsid, and Srivastava et al. (A. Srivastava et al., unpublished data) systematically replaced each of these tyrosine residues with phenylalanine. The consequence of these modifications is that the MOI required for the detection of transgene expression has been greatly reduced, both in cell culture and in several mouse models of transduction of cells in the liver and eye. This innovation is likely to greatly enhance the ability to increase transgene expression in several diseases to therapeutic levels.

One of the most attractive features of current AAV vectors is the continued expression of the transgene for prolonged periods of time. This is in spite of the extrachromosomal location of the vector. However, the infrequent integration of the vector means that transduction must occur in cells that either do not turn over or do so very slowly. Additionally, the rarity of integration reduces the likelihood of insertional mutagenesis, but the possibility does remain. Recent experience with the induction of leukemia in patients in two clinical trials who were successfully treated for severe combined immunodeficiency disease with retroviral vectors has heightened awareness of the problem ( 20). Although AAV vectors seem to be highly unlikely to cause such problems in postmitotic tissues, the issue remains of some concern. In contrast to the wild-type AAV genomes, recombinant AAV vector genomes do not integrate site specifically into chromosome 19 in human cells in vitro and have been shown to remain episomal in animal models in vivo. However, all previous studies were carried out with cells and tissues that are postmitotic. In hematopoietic stem cells, which must proliferate and differentiate to give rise to progenitor cells, recombinant AAV genomes would be lost in the absence of stable integration into chromosomal DNA. Srivastava and colleagues, using a murine bone marrow serial transplant model in vivo, documented the stable integration of the proviral genomes, and integration sites were localized to different mouse chromosomes (A. Srivastava et al., unpublished data). None of the integration sites was found to be in a transcribed gene or near a cellular oncogene. All animals monitored for up to 1 year exhibited no pathological abnormalities. Thus, an AAV proviral integration-induced risk of oncogenesis was not found in these studies.

One of the features of AAV is its ability to specifically integrate into chromosome 19q13.4 to establish latent infection. Current AAV vectors do not have this ability because they lack both the cis-active signal in P5 (IEE) and the trans-active proteins (Rep68 and Rep78) required for site-specific integration. The development of such a vector would enable the transduction of germ or progenitor cells and thus help to ensure long-term transgene expression in tissues where cell turnover is a consideration. If transduction were done ex vivo, it would theoretically be possible to clone cells in which site-specific integration had occurred in the absence of significant, additional random integration. Appropriate vector design would allow the rep gene to be expressed during transduction but not itself be incorporated. Two such vectors were described: one is an AAV/adenovirus hybrid ( 54), and the other is a bipartite AAV vector ( 78). In the latter case, Rep is expressed from one component, and the second component contains the cis-active IEE. Both systems are promising but in early stages of development.

Another area with great potential for improvement is the route of administration. This is particularly true for the use of AAV vectors in the central nervous system (CNS). Currently, vector administration requires an open neurosurgical procedure. This is true because of the blood-brain barrier and because of the desire to target specific areas of the CNS. The development of vectors that could achieve the required targeting specificity and the ability to penetrate the blood-brain barrier would greatly facilitate CNS gene therapy. A number of different methods have been suggested and tried with limited success to date.

Other possible advances include a better understanding of the host response and the requirements for the induction of tolerance and the development of more efficient systems for the production of AAV vectors ( 12).

DISCUSSION

Gene therapy requires three things: the identification of the defect at the molecular level, a correcting gene, and a way to introduce the gene into appropriate host cells (i.e., a vector). We now have a sophisticated understanding of the basic mechanisms of many genetic diseases, and many corresponding genes have been cloned and can be produced at high levels. The major hurdle to be surmounted is the development of adequate vectors. The wide variety of approaches that have been tried, many of which are still being studied, points to the challenge of developing effective vectors. The delivery methods that have been tried include purified DNA under hydrodynamic pressure, the shotgun approach using DNA adhering to gold particles, lipid-DNA complexes, and, finally, virus-based vectors. Although the first three methods have an inherent simplicity that is attractive, in practice, the efficiency of gene delivery and expression has been lower than what is required for therapeutic efficacy. Viruses, on the other hand, represent nature's vectors for the delivery and expression of exogenous genes in host cells. Here, the challenge is to maintain the efficiency of delivery and expression while minimizing any pathogenicity of the virus from which the vector was derived. In practice, the challenge has been significant. In the only clearly documented instance of therapeutic correction of an inborn error, the inherent oncogenic properties of the original virus (Moloney murine leukemia virus) were retained; 4 of 12 patients with X-linked severe combined immunodeficiency disease developed leukemia. In this experiment, bone marrow precursor cells were transduced and allowed to differentiate. Under these conditions, a vector that would integrate was needed.

Among current viral vectors, only those derived from retroviruses have the ability to integrate at a reasonable frequency; retroviruses require cell division for integration to occur, whereas lentiviruses and foamy viruses can enter the nucleus and integrate in nondividing cells. Lentiviral vectors carry the psychological burden of being derived from significant pathogens, but foamy viruses infect a high percentage of humans without having been implicated as the cause of disease. Although there are production challenges, very promising results have been obtained in a canine model of congenital granulomatosis. The overriding theoretical consideration is that retroviruses integrate at many sites in the human genome, so there is always the concern of insertional mutagenesis possibly causing oncogenesis.

AAV-2, and presumably other serotypes, has been reported to integrate at a specific site in the q arm of chromosome 19 (AAVS1). The frequency with which integration occurs in AAVS1 has been reported to be from 60 to 90%. This exceeds the frequency that has been observed, with the most successful vectors being derived from bacteriophage systems. However, current AAV vectors do not have this ability (they lack the sequences required both in trans and in cis), and integration, which has been observed to occur at a low frequency (10 −7), is random. As discussed above (Future Prospects), it is possible to design AAV vectors that can integrate in a site-specific manner; therefore, a DNA virus vector is possible.

AAV was initially considered as a vector by only a few laboratories. This undoubtedly reflected the lack of familiarity with the virus, since it is nonpathogenic and, thus, of interest only to those inherently interested in its distinctive biology. However, as noted above, with time, it has become among the most commonly used viral vectors. This is likely the consequence of several factors. First, almost all other viral vectors lead to an initial burst of transgene expression that commonly disappears after a relatively short time, measured in weeks. AAV transgene expression, on the other hand, frequently persists for years or the life time of the animal model. Second, other viral vectors have a greater capacity with which to insert the transgene(s). However, with time and clever engineering, it has been possible to insert originally very large transgenes into AAV vectors. Interestingly, it has also proved to be feasible to have split vectors in which one construct has slight sequence overlap with a second construct so that recombination after vector nuclear entry leads to the intact transgene product being expressed. Thus, the consequences of the small size of the AAV genome have been overcome to a large extent.

Another significant positive feature of AAV vectors is that they frequently do not elicit a deleterious immune response. This feature is dependent on the site of administration and the effective MOI of the vector used. Another factor is that AAV appears to be taken up poorly by dendritic cells. Finally, the small capacity of the genome has meant that no viral genes remain. In a parallel manner, the latest version of adenovirus vectors is the gutless vectors from which all viral genes have also been removed. Interestingly, the gutless adenovirus vectors still do not perform as well as AAV vectors in terms of expression persistence. It is tempting to speculate that the difference reflects the special structure of the AAV ITR, which could serve both as an insulator and to protect against cellular exonucleases.

Thus, AAV has become appreciated as a good vector for the transduction of postmitotic cells. At this time, retroviral vectors remain the vector of choice for the transduction of stem or progenitor cells despite the inherent concern of possible oncogenesis. These considerations apply for situations in which long-term transgene expression is desired. In cases such as immunization or vector-induced oncolysis, where expression at higher levels for relatively short periods of time is desirable, other viral vectors such as those derived from adenovirus and herpesvirus have more useful characteristics. What has become apparent is that different vectors have characteristics that are advantageous in specific cases. Thus, the notion of best vector depends on the question of what is best for what purpose.

FINAL COMMENTS

AAV remains a promising delivery system for the realization of the dream of gene therapy. It compares favorably to other viral vectors, especially when sustained transgene expression is desired. Although nonviral vector systems such as lipid-mediated vectors, hydrodynamic delivery, and the gene gun have been advocated and tried, to date, none have approached the efficacy of the viral delivery systems. Whether such development will occur remains unknown. AAV vectors have achieved some success, and it seems likely that some of the advances described above and others not yet envisioned will enable AAV to become an effective therapeutic agent.

Acknowledgments

We thank A. Srivastava for his helpful suggestions.

The work was supported in part by a grant from the USPHS, grant DK58327.

REFERENCES

# data[].attributes.attachment_link.title
Gene Therapy Using Adeno-Associated Virus Vectors

# data[].attributes.attachment_link.description
Summary: The unique life cycle of adeno-associated virus (AAV) and its ability to infect both nondividing and dividing cells with persistent expression have made it an attractive vector. An additional attractive feature of the wild-type virus is the lack ...

# data[].attributes.body
most of the trials in gene therapy have been aimed at the treatment of different cancers including lung, gynaecological, skin, urological, neurological and gastrointestinal tumors, as well as haematological malignancies and paediatric tumors. second most popular Cardiovascular gene therapy.
KK

# data[].attributes.attachment_link.description
Enter your email address below. If your address has been previously registered, you will receive an email with instructions on how to reset your password. If you don't receive an email, you should register as a new user

# data[].attributes.headline
Gene Therapy for Parkinson's Disease A.S.

# data[].attributes.subject
Gene Therapy for Parkinson's Disease A.S.

# data[].attributes.body
Clinical trials in Japan used AAV2 vectors to treat mild-late stage PD. In the six months after treatment, 6 patients showed improved motor function due to the injection of AAV2. There are further studies looking at injecting the AAV2 into the brain with promising results of curing the disease. Dopamine synthesis, one of the main issues causing PD, has been treated with another vector that codes for all of the necessary genes, and has been proven an effective treatment in primates.

# data[].attributes.attachment_link.readable_text
Abstract

Human gene therapy has made substantial progress since the initiation of the first clinical trials 20 years ago. Here, we summarized important applications of gene transfer protocols in the treatment of various human diseases using different viral vectors. Recent successful trials on the treatment of ocular diseases and inherited immune deficiencies are particularly encouraging and have raised hopes that human gene therapy as a standard treatment option will finally become a reality. While immune responses and insertional mutagenesis pose obstacles for this novel form of molecular medicine, continuous progress suggests that a wider range of diseases can be treated with gene therapy in the future.

Introduction

With advances in molecular cloning, gene transfer emerged in the late 1980s as a potentially revolutionary new form of molecular medicine, holding promise for novel therapies for many genetic and acquired diseases. After two decades of ups and downs, Science journal, in its December 18 th, 2009 issue, considered the "return of gene therapy" as one of the major scientific breakthroughs in 2009. Similarly, Time magazine, reporting on recent high-impact publications in clinical gene therapy, asked the question: "Is gene therapy finally ready for prime time?"

The concept of gene therapy for genetic disease is very simple: a functional copy of the defective gene is introduced to replace the missing function. In principle, this can be accomplished using ex vivo gene transfer to cells that had been removed from the patient. The genetically modified cells are subsequently re-introduced to the patient's body. Alternatively, the vector carrying the functional gene copy is directly injected into the body to achieve in vivo gene transfer. The first clinical trials were initiated in 1990 and published in 1995, summarizing ex vivo gene transfer to umbilical cord blood cells or to autologous T lymphocytes of children with severe combined immune deficiency (SCID) due to mutations in the adenosine deaminase (ADA) gene ( Blaese et al., 1995; Bordignon et al., 1995; Kohn et al., 1995). In these early studies, gene transfer protocols based on a murine retroviral vector were inefficient and unable to transduce hematopoietic stem cells (HSC), thereby limiting extent and duration of gene transfer. Since 1990, >1500 clinical trials have been approved worldwide utilizing viral and non-viral vectors and targeting various organs, cell types, and diseases.

The first decade of clinical gene therapy sought to establish safety of this novel treatment modality and to gain first experience in patients. In the late 1990s, superior gene transfer protocols and vectors were rapidly developed, and numerous successful treatments in small and large animal models of human diseases were reported. In the clinic, the dawn of the new millennium brought the first successes as well as setbacks. Immune responses and insertional mutagenesis emerged as main hurdles for gene therapy. A violent innate immune response to an intravenously delivered adenoviral vector caused the death of a patient with a rare metabolic disorder in 1999 ( Somia and Verma, 2000). In 2000, the first clearly successful gene therapy was reported. Ex vivo retroviral gene transfer of the γc-chain (common to several cytokine receptors) to autologous HSC (CD34⁺ bone marrow cells) () cured boys with X-linked SCID ( Cavazzana-Calvo et al., 2000). However, 4 of 10 children in this French trial and 1 of 10 children in a similar trial in the UK developed leukemia ( Hacein-Bey-Abina et al., 2008). Leukemic T cell clones showed integration near the LMO2 T cell oncogene ( Hacein-Bey-Abina et al., 2003). It is likely that the combination of the growth advantage of the gene-corrected T cells and the activation of LMO 2 was responsible for this outcome. Retroviral vectors integrate randomly into the host genome, but show a preference for transcriptionally active genes, and they contain sequences that are prone to activating nearby genes encoded by the host chromosome.

Successful Treatment of ADA-SCID

Unlike the experience with X-SCID, retroviral transduction of HSC continues to show high therapeutic efficacy in ADA-SCID without the development of leukemia ( Aiuti et al., 2009; Aiuti et al., 2002). Eight of ten children have essentially been cured in an Italian trial. For the past 8 years, these pediatric patients no longer required enzyme replacement therapy, they respond well to vaccination, and they live normal lives, attending school instead of living as "bubble" boys or girls. The protocol included mild bone marrow conditioning with busulfan, and traditional ADA enzyme replacement therapy was halted in order to increase engraftment of gene-modified cells and provide some growth advantage over non-modified cells, in which toxic purine metabolites accumulate. Gene engraftment of multipotent HSCs resulted in correction of multiple hematopoietic lineages in a polyclonal fashion. The ADA transgene was expressed in lymphocytes and red blood cells, thereby leading to systemic detoxification and restoration of the immune function.

Lentiviral Vectors -- Encouraging First Clinical Experiences

The newest viral vectors in clinical trial are HIV-based lentiviral vectors, in which all HIV genes have been deleted. Unlike murine gamma retroviral vectors, these retroviruses do not require cell division for gene transfer and integration into the host genome and can therefore transduce dividing and non-dividing target cells. Furthermore, higher levels of gene transfer to HSCs and transgene expression in hematopoietic progenitor cells have been achieved, thereby facilitating therapy. These features, for the first time, make gene therapy for globin disorders such as β-thalassemia or sickle cell disease a possibility ( May et al., 2000; Pawliuk et al., 2001). First results from an ongoing French gene therapy trial on lentiviral gene transfer for β-thalassemia are promising in that one treated patient has not required red blood cell transfusions for the past 16 months ( Kaiser, 2009). Again, gene transfer was performed ex vivo to HSCs. However, a concern has emerged as well: in spite of very low-efficiency of gene transfer, approximately 10% of the patient's blood cells contain viral integrants in the HMGA2 gene, apparently resulting in increased gene expression and a growth advantage. Thus far, this does not seem to have caused adverse effects in the patient. Interestingly, in an earlier trial, which showed transient efficacy in two subjects with a rare immune deficiency called chronic granulomatous disease (CGD), murine retroviral HSC gene transfer caused expansion of myeloid clones ( Ott et al., 2006). CGD is characterized by lack of functional phagocytic cells due to mutations in genes required for oxidase function in these cells, thereby reducing the immune system's ability to fend off microbial pathogens. Viral integration upregulated expression of genes that seemed to favor proliferation of cells that are beneficial to therapy. However, efficacy was subsequently lost because of transgene silencing and activation of endogenous EVI1 gene, causing genomic instability ( Stein et al., 2010).

Any of these events linked to changes in endogenous gene expression upon viral integration were unexpected, and therefore underscore that much remains to be learned about the effects of insertion of viral sequences and therapeutic expression cassettes into host genomes. In the long-run, development of site-specific integration systems is desirable.

Nonetheless, the first successful use of lentiviral gene transfer to HSC was recently reported for X-linked adrenoleukodystrophy (ADL), a devastating lipid storage disorder in boys that results in demyelination of neurons in the brain. Gene transfer halted the progressive brain damage in two 7-year old patients ( Cartier et al., 2009). It is possible that benefits from the use of integrating vectors in many cases and diseases will out-weigh risks associated with insertional mutagenesis, in particular as vectors are being developed with reduced impact on cellular gene expression and as these risks are being better understood. Furthermore, non-integrating lentiviral vectors have also been developed recently, but their long-term safety and efficacy remains to be evaluated.

Adeno-associated Viral (AAV) Vectors for the Treatment of Inherited Blindness

AAV vectors are derived from a non-pathogenic parent virus with a single-stranded DNA genome. High efficiency of in vivo gene transfer to numerous cell types, development of a large arsenal of viral capsids with different tropisms, and low innate/inflammatory responses to viral particles have made these vectors highly attractive. Numerous investigations have yielded impressive results on the correction of various genetic diseases in mice, dogs, non-human primates, and other animal models ( Warrington and Herzog, 2006). For example, hepatic coagulation factor IX gene transfer resulted in greater than eight years of stable correction of hemophilia B in canines and was also shown to induce immune tolerance to factor IX and several other therapeutic proteins ( LoDuca et al., 2009; Niemeyer et al., 2009). However, similar therapeutic expression in humans was not achieved because of pre-existing antibodies against the capsid of this particular serotype (AAV2), or was transient because of a CD8⁺ T cell response to the viral capsid ( Manno et al., 2006).

In a gene therapy trial for Parkinson's disease (PD) with AAV2 vectors, some clinical efficacy was observed ( Kaplitt et al., 2007). At the 2009 annual meeting of the American Society of Gene and Cell Therapy, Muramatsu and colleagues reported results from a more recent trial in Japan. Six patients with mild- to late-stage PD treated with AAV2 vectors showed marked improvement in motor functions six-months post-treatment. In ongoing clinical trials, AAV gene transfer to the brains of patients with Parkinson's disease has shown promising results. Partial restoration of dopamine synthesis combined with L-dopa administration can ameliorate disease. Furthermore, a tricistronic lentiviral vector encoding all genes required for dopamine synthesis has been successfully tested in nonhuman primates ( Jarraya et al., 2009).

In 2008, three groups (two in the US and one in the UK) reported success in patients using the AAV2 vector ( Bainbridge et al., 2008; Cideciyan et al., 2008; Maguire et al., 2008). Local (subretinal, an immune privileged site) administration of a vector expressing RPE65 (retinal pigment epithelium-specific 65 kD protein) led to gain of light sensitivity and, in some cases, of vision, in patients with Leber's congenital amaurosis (LCA). This rare form of inherited blindness is caused by progressive loss of rod and cone function due to the lack of the RPE65 isomerase, which is required to form light sensitive pigment. Proof of concept with this gene therapy approach had initially been achieved in animals in 2001, when vision was restored in a Briard dog, named Lancelot ( Acland et al., 2001). Gain of light sensitivity was documented at the site of retinal gene transfer (), and patients gained vision as evidenced by behavior correlates such as the ability to walk through a maze ( Cideciyan et al., 2008). A one-year follow-up showed no diminution in any of the clinical parameters ( Cideciyan et al., 2009). A subsequent trial in children showed that the extent of visual improvement achieved by the gene therapy is age-dependent ( Maguire et al., 2009). Earlier intervention, when degeneration of photoreceptors has progressed less, results in substantial gains in ambulatory vision. Most impressively, four children (8-11 years old) are now able to play sports and attend school without the use of learning aids. Another recent study, demonstrating correction of color-blindness in squirrel monkeys by ocular therapy with an AAV2 vector (), provides a glimpse of future therapies to come ( Mancuso et al., 2009).

More Advances in the Foreseeable Future

There are many more promising gene therapy protocols in clinical trials or in the pre-clinical pipelines. Existing protocols that encountered obstacles are being modified to overcome identified hurdles. For example, AAV vector capsids are being engineered to be more resistant to neutralizing antibodies in the human population, to transduce target cells more efficiently, and to show enhanced tropism to specific tissues ( Zhong et al., 2008). One exciting development is the ability to transfer a gene to cardiac and multiple skeletal muscles, which holds promise for the treatment of muscular dystrophies and lysosomal storage disorders ( Asokan et al., 2009). Some gene transfer protocols may include transient immune suppression or modulation to prevent adaptive responses against capsid or transgene derived antigen ( Sack and Herzog, 2009). Such approaches are not unlike cell and organ transplantation or protein replacement therapies, which are also often rejected by the immune system. Future vectors may also include microRNA-regulated expression cassettes to detarget expression from professional, bone marrow-derived antigen presenting cells ( Brown et al., 2006). With regard to degenerative muscle disorders, a recent study accomplished robust improvements of muscle growth and strength in nonhuman primates using AAV-mediated follistatin gene transfer ( Kota et al., 2009).

In summary, human gene therapy has become a reality, clinical gene therapy is here to stay, and more successful protocols are expected to emerge in the near future. Treatment of ocular diseases and of inherited immune deficiencies are particularly successful. The most severe hurdles that have been encountered are immunemediated rejection of the therapy or complications arising from insertional mutagenesis (especially following gene transfer to stem cells). Success in a wider range of diseases will depend on solutions to these problems.

Contributor Information

Roland W. Herzog, Department of Pediatrics, University of Florida College of Medicine, 2033 Mowry Road, Gainesville, Florida 32610, USA.

Ou Cao, Department of Pediatrics, University of Florida College of Medicine, 2033 Mowry Road, Gainesville, Florida 32610, USA.

Arun Srivastava, Departments of Pediatrics and Molecular Genetics & Microbiology, University of Florida College of Medicine, 2033 Mowry Road, Gainesville, Florida 32610, USA.

# data[].attributes.attachment_link.title
Two Decades of Clinical Gene Therapy - Success Is Finally Mounting

# data[].attributes.attachment_link.description
Human gene therapy has made substantial progress since the initiation of the first clinical trials 20 years ago. Here, we summarized important applications of gene transfer protocols in the treatment of various human diseases using different viral vectors.

# data[].attributes.body
At the start of the process a carrier called a vector is genetically engineered to deliver an altered gene. A virus is often used because they can deliver the new gene by infecting the targeted cell. The viruses have to be modified so they can't cause disease when used to transport. The vector is then injected via IV directly into a specific tissue in the body, where it is taken up by individual cells. another way to incert the new genes, "a sample of the patient's cells can be removed and exposed to the vector in a laboratory setting".(National Library of Medicine) If the treatment is successful, the new gene delivered by the vector will make a functioning protein.

# data[].attributes.attachment_link.description
The vector can be injected or given intravenously (by IV) directly into a specific tissue in the body, where it is taken up by individual cells. Alternately, a sample of the patient's cells can be removed and exposed to the vector in a laboratory setting.

# data[].attributes.body
CRISPR is a unique organization of DNA sequences which could be found in genomes of bacteria and other microorganisms. CRISPR is also a crucial component of the immune system. The way CRISPR works is that it protects bacteria from repeated viral attacks by producing CRISPR RNA which is just copied DNA that is then transformed into RNA and then cut into little pieces call CRISPR RNA. Then the CRISPR RNA targets and attacks viruses that are harmful
RC

# data[].attributes.attachment_link.readable_text
Have you heard? A revolution has seized the scientific community. Within only a few years, research labs worldwide have adopted a new technology that facilitates making specific changes in the DNA of humans, other animals, and plants. Compared to previous techniques for modifying DNA, this new approach is much faster and easier. This technology is referred to as "CRISPR," and it has changed not only the way basic research is conducted, but also the way we can now think about treating diseases [1,2].

What is CRISPR

CRISPR is an acronym for Clustered Regularly Interspaced Short Palindromic Repeat. This name refers to the unique organization of short, partially palindromic repeated DNA sequences found in the genomes of bacteria and other microorganisms. While seemingly innocuous, CRISPR sequences are a crucial component of the immune systems [3] of these simple life forms. The immune system is responsible for protecting an organism's health and well-being. Just like us, bacterial cells can be invaded by viruses, which are small, infectious agents. If a viral infection threatens a bacterial cell, the CRISPR immune system can thwart the attack by destroying the genome of the invading virus [4]. The genome of the virus includes genetic material that is necessary for the virus to continue replicating. Thus, by destroying the viral genome, the CRISPR immune system protects bacteria from ongoing viral infection.

How does it work?
Figure 1 ~ The steps of CRISPR-mediated immunity. CRISPRs are regions in the bacterial genome that help defend against invading viruses. These regions are composed of short DNA repeats (black diamonds) and spacers (colored boxes). When a previously unseen virus infects a bacterium, a new spacer derived from the virus is incorporated amongst existing spacers. The CRISPR sequence is transcribed and processed to generate short CRISPR RNA molecules. The CRISPR RNA associates with and guides bacterial molecular machinery to a matching target sequence in the invading virus. The molecular machinery cuts up and destroys the invading viral genome. Figure adapted from Molecular Cell 54, April 24, 2014 [5].
Interspersed between the short DNA repeats of bacterial CRISPRs are similarly short variable sequences called spacers (FIGURE 1). These spacers are derived from DNA of viruses that have previously attacked the host bacterium [3]. Hence, spacers serve as a 'genetic memory' of previous infections. If another infection by the same virus should occur, the CRISPR defense system will cut up any viral DNA sequence matching the spacer sequence and thus protect the bacterium from viral attack. If a previously unseen virus attacks, a new spacer is made and added to the chain of spacers and repeats.

The CRISPR immune system works to protect bacteria from repeated viral attack via three basic steps [5]:

Step 1) Adaptation - DNA from an invading virus is processed into short segments that are inserted into the CRISPR sequence as new spacers.

Step 2) Production of CRISPR RNA - CRISPR repeats and spacers in the bacterial DNA undergo transcription, the process of copying DNA into RNA (ribonucleic acid). Unlike the double-chain helix structure of DNA, the resulting RNA is a single-chain molecule. This RNA chain is cut into short pieces called CRISPR RNAs.

Step 3) Targeting - CRISPR RNAs guide bacterial molecular machinery to destroy the viral material. Because CRISPR RNA sequences are copied from the viral DNA sequences acquired during adaptation, they are exact matches to the viral genome and thus serve as excellent guides.

The specificity of CRISPR-based immunity in recognizing and destroying invading viruses is not just useful for bacteria. Creative applications of this primitive yet elegant defense system have emerged in disciplines as diverse as industry, basic research, and medicine.

What are some applications of the CRISPR system?
In Industry
The inherent functions of the CRISPR system are advantageous for industrial processes that utilize bacterial cultures. CRISPR-based immunity can be employed to make these cultures more resistant to viral attack, which would otherwise impede productivity. In fact, the original discovery of CRISPR immunity came from researchers at Danisco, a company in the food production industry [2,3]. Danisco scientists were studying a bacterium called Streptococcus thermophilus, which is used to make yogurts and cheeses. Certain viruses can infect this bacterium and damage the quality or quantity of the food. It was discovered that CRISPR sequences equipped S. thermophilus with immunity against such viral attack. Expanding beyond S. thermophilus to other useful bacteria, manufacturers can apply the same principles to improve culture sustainability and lifespan.

Beyond applications encompassing bacterial immune defenses, scientists have learned how to harness CRISPR technology in the lab [6] to make precise changes in the genes of organisms as diverse as fruit flies, fish, mice, plants and even human cells. Genes are defined by their specific sequences, which provide instructions on how to build and maintain an organism's cells. A change in the sequence of even one gene can significantly affect the biology of the cell and in turn may affect the health of an organism. CRISPR techniques allow scientists to modify specific genes while sparing all others, thus clarifying the association between a given gene and its consequence to the organism.

Rather than relying on bacteria to generate CRISPR RNAs, scientists first design and synthesize short RNA molecules that match a specific DNA sequence-for example, in a human cell. Then, like in the targeting step of the bacterial system, this 'guide RNA' shuttles molecular machinery to the intended DNA target. Once localized to the DNA region of interest, the molecular machinery can silence a gene or even change the sequence of a gene (Figure 2)! This type of gene editing can be likened to editing a sentence with a word processor to delete words or correct spelling mistakes. One important application of such technology is to facilitate making animal models with precise genetic changes to study the progress and treatment of human diseases.
Figure 2 ~ Gene silencing and editing with CRISPR. Guide RNA designed to match the DNA region of interest directs molecular machinery to cut both strands of the targeted DNA. During gene silencing, the cell attempts to repair the broken DNA, but often does so with errors that disrupt the gene-effectively silencing it. For gene editing, a repair template with a specified change in sequence is added to the cell and incorporated into the DNA during the repair process. The targeted DNA is now altered to carry this new sequence. In Medicine
With early successes in the lab, many are looking toward medical applications of CRISPR technology. One application is for the treatment of genetic diseases. The first evidence that CRISPR can be used to correct a mutant gene and reverse disease symptoms in a living animal was published earlier this year [7]. By replacing the mutant form of a gene with its correct sequence in adult mice, researchers demonstrated a cure for a rare liver disorder that could be achieved with a single treatment. In addition to treating heritable diseases, CRISPR can be used in the realm of infectious diseases, possibly providing a way to make more specific antibiotics that target only disease-causing bacterial strains while sparing beneficial bacteria [8]. A recent SITN Waves article discusses how this technique was also used to make white blood cells resistant to HIV infection [9].

The Future of CRISPR

Of course, any new technology takes some time to understand and perfect. It will be important to verify that a particular guide RNA is specific for its target gene, so that the CRISPR system does not mistakenly attack other genes. It will also be important to find a way to deliver CRISPR therapies into the body before they can become widely used in medicine. Although a lot remains to be discovered, there is no doubt that CRISPR has become a valuable tool in research. In fact, there is enough excitement in the field to warrant the launch of several Biotech start-ups that hope to use CRISPR-inspired technology to treat human diseases [8].
Ekaterina Pak is a Ph.D. student in the Biological and Biomedical Sciences program at Harvard Medical School.
References:

1. Palca, J. A CRISPR way to fix faulty genes. (26 June 2014) NPR < http://www.npr.org/blogs/health/2014/06/26/325213397/a-crispr-way-to-fix-faulty-genes> [29 June 2014]

2. Pennisi, E. The CRISPR Craze. (2013) Science, 341 (6148): 833-836.

3. Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., Romero, D.A., and Horvath, P. (2007). CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709-1712.

4. Brouns, S.J., Jore, M.M., Lundgren, M., Westra, E.R., Slijkhuis, R.J., Snijders, A.P., Dickman, M.J., Makarova, K.S., Koonin, E.V., and van der Oost, J. (2008). Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321, 960-964.

5. Barrangou, R. and Marraffini, L. CRISPR-Cas Systems: Prokaryotes Upgrade to Adaptive Immunity (2014). Molecular Cell 54, 234-244.

6. Jinkek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.(2012) 337(6096):816-21.

7. CRISPR reverses disease symptoms in living animals for first time. (31 March 2014). Genetic Engineering and Biotechnology News. < http://www.genengnews.com/gen-news-highlights/crispr-reverses-disease-symptoms-in-living-animals-for-first-time/81249682/> [27 July 2014]

8. Pollack, A. A powerful new way to edit DNA. (3 March 2014). NYTimes < http://www.nytimes.com/2014/03/04/health/a-powerful-new-way-to-edit-dna.html?_r=0> [16 July 2014]

9. Gene editing technique allows for HIV resistance? < http://sitn.hms.harvard.edu/flash/waves/2014/gene-editing-technique-allows-for-hiv-resistance/> [13 June 2014]

# data[].attributes.attachment_link.title
CRISPR: A game-changing genetic engineering technique - Science in the News

# data[].attributes.attachment_link.description
Have you heard? A revolution has seized the scientific community. Within only a few years, research labs worldwide have adopted a new technology that facilitates making specific changes in the DNA of humans, other animals, and plants. Compared to previous techniques for modifying DNA, this new approach is much faster and easier.

# data[].attributes.body
Gene therapy is a treatment that involves altering the genes with altered DNA to stop disease.
KK

# data[].attributes.attachment_link.description
Learn about gene therapy, which replaces a faulty gene or adds a new gene in an attempt to cure disease or improve your body's ability to fight disease.