https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p Presented by Shesha Taylor, Spencer Abssy, Aled Blundell, Tej Patel en-us 2022-12-03 18:15:22 UTC 2025-10-27 20:26:56 UTC hello@padlet.com aledblundell https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2407701587 Paper title: Efficient spatially targeted gene editing using a near-infrared activatable protein-conjugated nanoparticle for brain applications ]]> 2022-12-03 21:58:55 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2407701587 spencerabssy https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2408102899 Rebelo et al. created an up conversion nanoparticle (UCNP). This nanoparticle has four major components the UCNP body (core + shell), a photocleavable linker, hydroxychloroquine attachment, and Cre recombinase. Specific properties within the UCNP core allows for the upconversion of NIR light to blue light. ]]> 2022-12-04 16:10:13 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2408102899 spencerabssy https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2408103525 UCNP delivery can be defined in the four stages: (1) Clathrin-mediated endocytosis, (2) Endosomal escape, (3) NIR-dependent release, and (4) Nucleus migration.]]> 2022-12-04 16:11:22 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2408103525 spencerabssy https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2408106420 Ultraviolet (UV) light has been seen to damage target DNA and induce in vitro cellular death. UV and visible light (VIS) waves lack effective penetrance, because of their high energy states. Instead of penetrating, they are mostly absorbed by biological tissue (Wang et al., 2013). Even though UV/VIS light lack penetrance, they contain enough energy to break photocleavable bonds. This is a function that NIR light cannot perform. NIR light exists at a lower energy state than UV or VIS light. This allows for a significant increase in tissue penetrance depth, but not the cleavage of photocleavable bonds (Shi et al., 2019). Through using upconversion of NIR light to blue light, we can avoid the penetrative drawbacks of VIS light and the energy drawbacks associated to NIR light. ]]> 2022-12-04 16:16:41 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2408106420 spencerabssy https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2408115619 Controlling in vivo gene expression with precision and spatial resolution, with the capacity to evaluate activity in the edited cells, has not been seen. ]]> 2022-12-04 16:32:55 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2408115619 spencerabssy https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2408117956 Light activatable Cre recombinase systems have been commonly used to modulate neuron activity with great precision and temporal agility. However, these effects are highly invasive and seen primarily in vitro. Edwards et al. demonstrate this limitation through optogenetic studies using blue light as an activator. Stereotaxic administration was the only consistent methods for stimulating light responsive elements that regulate the Cre recombinase system. UV/blue light, which has limited tissue penetration ability. (Edwards, 2009)]]> 2022-12-04 16:36:52 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2408117956 spencerabssy https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2408118365 Morals et al. attempted to address UV light based limitations by developing a nanoparticle that delivers Cre recombinase to target sites after near-infrared (NIR) light activation. They developed a gold nanoparticle shell, which participated in covalent binding with Cre recombinase. This method of Cre immobilization resulted in low control over protein delivery, cellular uptake, and a 20 % cell recombination rate in vitro. This study did not demonstrate in vivo gene editing capabilities. (Morales et al., 2018)]]> 2022-12-04 16:37:32 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2408118365 spencerabssy https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2408127120

Stereotaxic administration of UCNP Cre recombinase systems (Cre-UCNPs) in the subventricular zone (SVZ) demonstrated recombination of a loxP cassette in brain cells of transgenic mice after NIR laser exposure  

Stereotaxic administration of Cre-UCNPs in the ventral tegmental area (VTA) with NIR activation demonstrated control over reward processes and behavioral reinforcement via blue light CRE-dependent channelrhodopsin-2 (CHR2) activation 

Demonstrated spatial resolution after gene editing with Cre-UCNPs delivered via a non-invasive administration route (intranasal installation)

]]> 2022-12-04 16:52:58 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2408127120 tejrainn https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2408437478 Use of CRISPR-Cas9 in conjuction with UCNP ]]> 2022-12-05 01:41:05 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2408437478 sheshataylor1 https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411338830 An overview and explanation of the experiments.]]> 2022-12-06 22:08:11 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411338830 sheshataylor1 https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411406229

Chen, Shuo et al. “Near-infrared deep brain stimulation via upconversion nanoparticle-mediated optogenetics.” Science (New York, N.Y.) vol. 359,6376 (2018): 679-684.

Dichter, Gabriel S et al. “Reward circuitry dysfunction in psychiatric and neurodevelopmental disorders and genetic syndromes: animal models and clinical findings.” Journal of neurodevelopmental disorders vol. 4,1:19 (2012).

Ditmangklo, B., Boonlua, C., Suparpprom, C., & Vilaivan, T. (2013). Reductive alkylation and sequential reductive alkylation-click chemistry for on-solid-support modification of pyrrolidinyl peptide nucleic acid. Bioconjugate chemistry, 24(4), 614–625.

Edwards, W. F., Young, D. D. & Deiters, A. Light-Activated Cre Recombinase as a Tool for the Spatial and Temporal Control of Gene Function in Mammalian Cells. Acs Chem. Biol. 4, 441–445 (2009).

Forni, Paolo E et al. “High levels of Cre expression in neuronal progenitors cause defects in brain development leading to microencephaly and hydrocephaly.” The Journal of neuroscience: the official journal of the Society for Neuroscience vol. 26,37 (2006): 9593-602.

Geibel, C., Theiner, J., Wolter, M., Kramer, M., Lindner, W., & Lämmerhofer, M. (2021). Controllable organosilane monolayer density of surface bonding using silatranes for thiol functionalization of silica particles for liquid chromatography and validation of microanalytical method for elemental composition determination. Journal of chromatography. A, 1653, 462418.

Guo, Shaowei et al. “Intranasal Delivery of Mesenchymal Stem Cell Derived Exosomes Loaded with Phosphatase and Tensin Homolog siRNA Repairs Complete Spinal Cord Injury.” ACS nano vol. 13,9 (2019): 10015-10028.

Kawano, Fuun et al. “A photoactivatable Cre-loxP recombination system for optogenetic genome engineering.” Nature chemical biology vol. 12,12 (2016): 1059-1064.

Kennedy, Matthew J et al. “Rapid blue-light-mediated induction of protein interactions in living cells.” Nature methods vol. 7,12 (2010): 973-5.

Madisen, Linda et al. “A robust and high-throughput Cre reporting and characterization system for the whole mouse brain.” Nature neuroscience vol. 13,1 (2010): 133-40.

Morales, Demosthenes P et al. “Light-Triggered Genome Editing: Cre Recombinase Mediated Gene Editing with Near-Infrared Light.” Small (Weinheim an der Bergstrasse, Germany) vol. 14,30 (2018): e1800543.

Nakajima, Mitsunari et al. “Psychiatric disorder-related abnormal behavior and habenulointerpeduncular pathway defects in Wnt1-cre and Wnt1-GAL4 double transgenic mice.” Journal of neurochemistry vol. 124,2 (2013): 241-9.

Pan, Yongchun, et al. “Near-Infrared Upconversion–Activated CRISPR-Cas9 System: A Remote-Controlled Gene Editing Platform.” Science Advances vol. 5,4 (2019). 

Rebelo, Catarina, et al. “Efficient Spatially Targeted Gene Editing Using a Near-Infrared Activatable Protein-Conjugated Nanoparticle for Brain Applications.” Nature Communications, vol. 13, no. 1, 16 July 2022, eg.uc.pt/handle/10316/101264, 10.1038/s41467-022-31791-6. Accessed 13 Nov. 2022.

Smith, Samuel A., et al. “The Endosomal Escape of Nanoparticles: Toward More Efficient Cellular Delivery.” Bioconjugate Chemistry, vol. 30, no. 2, 19 Nov. 2018, pp. 263–272, 10.1021/acs.bioconjchem.8b00732.

Sukumar, Uday K et al. “Intranasal delivery of targeted polyfunctional gold-iron oxide nanoparticles loaded with therapeutic microRNAs for combined theranostic multimodality imaging and presensitization of glioblastoma to temozolomide.” Biomaterials vol. 218 (2019): 119342.

Wang, Ye-Fu, et al. “Nd³⁺-Sensitized Upconversion Nanophosphors: Efficient in Vivo Bioimaging Probes with Minimized Heating Effect.” ACS Nano, vol. 7, no. 8, 23 July 2013

Zhang, Wei et al. “Optogenetic control with a photocleavable protein, PhoCl.” Nature methods vol. 14,4 (2017): 391-394.

Zuris, John A et al. “Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo.” Nature biotechnology vol. 33,1 (2015): 73-80.

Pickens, C. J., Johnson, S. N., Pressnall, M. M., Leon, M. A., & Berkland, C. J. (2018). Practical Considerations, Challenges, and Limitations of Bioconjugation via Azide-Alkyne Cycloaddition. Bioconjugate chemistry, 29(3), 686–701.

]]> 2022-12-06 23:56:47 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411406229 spencerabssy https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411407367 In Clathrin-mediated endocytosis, the UCNP system is taken up into a plasma membrane vesicle. This vesicle allows entry into cellular cytoplasm, but traps nanoparticles inside. this can lead to UCNP degradation through lysosomal interactions, disallowing the UCNP delivery to target locations. (Smith et al., 2018) ]]> 2022-12-06 23:58:18 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411407367 sheshataylor1 https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411407502 Introduction: Spencer Abssy
Methods and Results: Aled Blundell and Shesha Taylor
Discussion: Tej Patel

We would like to thank TA Filip and professor Naomi Levy-Strumpf for their support throughout this project!]]> 2022-12-06 23:58:28 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411407502 spencerabssy https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411408223 Endosomal escape occurs to release UCNPs from vesicles. Although this mechanism has not fully been elucidated, it is thought that endosomal maturation processes cause breaches in lipid bilayer. (Dowdy et al., 2022). This breach allows for the subsequent escape of UCNPs from the vesicle. HCQ treatment is thought to decrease the cellular activity of lysosomes through LAMP1+ tags. 

]]> 2022-12-06 23:59:26 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411408223 spencerabssy https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411408900 NIR light exposure facilitates the destruction of the photocleavable bond between the UCNP shell and Cre recombinase, through upconversion of NIR light to blue light. ]]> 2022-12-07 00:00:21 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411408900 spencerabssy https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411410100 2022-12-07 00:01:59 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411410100 sheshataylor1 https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411412544

Selectivity between Cre-PCL-azide and HCQ-amine coupling allows for precise ratios of Cre-PCL and HCQ to be added, based on concentrations of azide or amine bearing organosilanes addded

• Organosilanes used as the backbone because they are well characterized, bioorthogonal coupling agents that act as an interface between organic and inorganic compounds

• This “Click chemistry” approach is so robust, 2022 Nobel prize in chemistry awarded to inventors (Bertozzi, Sharpless, and Meldal)

]]> 2022-12-07 00:05:13 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411412544 sheshataylor1 https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411413109 2022-12-07 00:06:00 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411413109 sheshataylor1 https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411416782 According to graphs 1 and 2, the amount of protein released after NIR irradition was huge initially but slowed as its concentration decreased. Cellular internalization of the Cre-UCNPs was also relatively quickly, taking between 2-4 hours. It was dependent on the NP concentration and presence of the HCQ. There was no measurable cytotoxicity observed for concentrations less than or equal to 50 μg/mL and irradiation times less than 30 min. Under these conditions, irradiated cells showed no heat shock response and no double-stranded DNA breaks.]]> 2022-12-07 00:10:37 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411416782 sheshataylor1 https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411417464 To identify the pathway through which Cre-UCNP penetrated cells, fibroblasts were transfected with fluorescently labeled Cre-UCNPs in the presence of endocytosis inhibitors. Particle internalization was assessed via flow cytometry. The results showed that the main internalization mechanism was mediated by clathrin-mediated endocytosis.]]> 2022-12-07 00:11:30 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411417464 sheshataylor1 https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411417810 Cells were exposed to Cre-UCNPs and monitored by confocal microscopy to determine the staining of Cre and LAMP1. After exposure to NPs, co-localization of Cre-UCNPs with stained LAMP1 foci was reduced compared to Cre-UCNPs without HCQ. This may indicate disrupted lysosome biogenesis which could be because HCQ impairs autophagosome fusion with lysosomes. To determine whether Cre-UCNPs with HCQ induced endolysosomal compartment disruption, they monitored galectin-9 by immunofluorescence, which is a sensitive sensor of membrane damage. Cells exposed to Cre-UCNPs with HCQ showed an increased number of galectin-9 foci compared to those treated with Cre-UCNPs without HCQ.]]> 2022-12-07 00:11:57 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411417810 sheshataylor1 https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411427644 2022-12-07 00:23:30 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411427644 sheshataylor1 https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411429590 2022-12-07 00:25:40 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411429590 sheshataylor1 https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411430700 2022-12-07 00:26:38 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411430700 sheshataylor1 https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411445770 To show the potential for gene editing through Cre-UCNPs in brain cells, they targeted SVZ cells since they are an important in the adult mammalian neurogenic niche. Cells were isolated from Rosa26-YFP transgenic mice, which contain a LoxP-Stop-LoxP-YFP sequence cassette inserted into the Gt(ROSA)26Sor locus . ]]> 2022-12-07 00:41:28 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411445770 tejrainn https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411447863

Authors overcame the issues in previous studies (Zhang et al., 2017; Kawano et al., 2016; Kennedy et al., 2010) where the systems were based on the constitutive expression of Cre and relied on the activation of UV/blue light which has limited tissue penetration and cellular transfection ability. 

• Authors were able to gain higher control over protein delivery and observed improvements in cellular uptake and intracellular delivery in comparison to previous Cre recombinase formulations. 
• Their UCNP formulation allowed transient expression of Cre recombinase with 125-fold higher efficiency than commercially available products and did not require further protein engineering to facilitate cellular uptake (Zuris et al., 2015).

Authors enhanced the viral delivery of Cre-expressing constructs methods through the development of their UCNP formulation, which is a combined alternative with significantly reduced safety problems in the rhodopsin delivery method and effective NIR-mediated activation.

Several previous works have reported the nonspecific and potentially toxic effects of Cre recombinase in mice in several organs, including the brain, in addition to the physiological complications of the Cre-driver line mice (Forni et al., 2006; Nakajima et al., 2013).

• In turn, this UCNP formulation is a hopeful system that is capable of alleviating the existing side effects of the Cre recombinase technique to conduct successful recombinase activity.

Authors have incorporated a non-invasive route of delivery to make an easy administration route, reduce pain and/or discomfort, and for a rapid recovery period. 

• They performed targeted gene editing through intranasal instillation where the nanoparticles administered nasally can reach the brain, which has been established by previous studies (Guo et al., 2019; Sukumar et al., 2019).

]]> 2022-12-07 00:43:42 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411447863 tejrainn https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411448594

Rebelo et al. have performed a comprehensive set of experiments that developed a delivery system for gene-editing enzymes that is able to effectively facilitate endolysosomal escape and release within deep biological tissue following exposure to transcranial NIR light with great spatial resolution. 

The authors demonstrated the effectiveness of the UCNP formulation in vivo of the mouse brain while successfully gene editing in neurogenic niches, which are specialized microenvironments that regulate neural progenitor cell (NPC) activity. They also activated mechanisms of reward and reinforcement in the VTA using NIR activation to genetically expose an optogenetic actuator (ChR2), followed by blue light activation.

]]> 2022-12-07 00:44:28 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411448594 tejrainn https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411455351 NIR capable of penetrating deep biological tissue for research and therapeutic purposes.]]> 2022-12-07 00:52:22 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411455351 tejrainn https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411459275

Authors did not test the biological modulation of brain cell activity via chemogenetics. While their focus was on optogenetics, the authors hint at how chemogenetics would have been a more non-invasive approach and that they may have been able to incorporate transcription factor genes into the brain and subsequently measure their impact at the functional level.

The non-invasive route of delivery, intranasal instillation, wasn’t as well emphasized in the paper and only in the supplementary data, suggesting that the results of that experiment weren’t as successful.

The entire UCNP formulation was very complex and thus very difficult, time consuming, and expensive in terms of reproducibility.

]]> 2022-12-07 00:56:24 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411459275 aledblundell https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411463469 Culturing Cre-reporter fibroblast cell lines for 48h after Cre-UCNP delivery and NIR light exposure produced a near 100% recombination efficiency]]> 2022-12-07 01:01:21 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411463469 aledblundell https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411467144 Cre-UCNP recombination efficiency without NIR light exposure in fibroblast cell cultures is ~20%.  With NIR light exposure, recombination efficiency is dramatically higher (~100%)]]> 2022-12-07 01:05:22 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411467144 tejrainn https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411468398

In the absence of light activation, only Cre-UCNPs with HCQ, were able to reliably facilitate endolysosomal escape similar to the disruptive ability of soluble HCQ. 

The synergistic effects of the immobilized HCQ molecules allows for very low concentrations of immobilized HCQ in NPs can be as equally efficient as high concentrations of soluble HCQ (60-fold higher).

• The authors harnessed the capacity to control the activity of genes with precise spatial control, using the UCNP formulation that can be externally activated using NIR light. 
• Unique because previous studies have not been able to acquire this level of control using NIR light-triggerable NP formulations.

The authors used optogenetics to monitor the activity of the edited cells and the level of gene editing in the brain using behavioural tests.

• Ability to control the activity of genes with precise spatial control, using a Cre-based NP formulation that can be externally triggered using NIR light
  • First to show Cre-UCNPs ability to induce in vitro cell recombination at the single-cell level, with spatial resolution (Morales et al., 2018)
  • Showed that Cre-UCNPs can induce in vivo cell recombination, with spatial resolution at the level of the cerebral hemisphere 
  • Exhibited that the spatial control achieved by Cre-UCNPs in the VTA region was significantly higher than with an AAV5 virus

]]> 2022-12-07 01:06:43 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411468398 aledblundell https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411469070 2022-12-07 01:07:29 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411469070 tejrainn https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411472893 Differences between chemogenetics and optogenetics in a mouse experiment]]> 2022-12-07 01:11:33 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411472893 tejrainn https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411474492

This development is extremely useful as it allows for momentary expression of Cre recombinase with significantly greater efficiency than current commercially available methods and can be utilized for the treatment of psychiatric reward-pathway impairments (substance-use, obsessive-compulsive disorders), neurodevelopmental disorders (schizophrenia, autism spectrum disorders), and genetic syndromes (fragile X syndrome, Angelman syndrome) (Dichter et al., 2012). 

This UCNPs formulation has the potential to be used in the stimulation of deep brain neurons which is in line with recent works in this field. 

]]> 2022-12-07 01:13:17 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411474492 aledblundell https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411477057 Fibroblast Cre-reporter cells administered Cre-UCNPS without surface bound HCQ and exposed to NIR light showed only ~2.6% recombination, while Cre-UCNPs with HCQ demonstrated near 100% recombination.]]> 2022-12-07 01:15:53 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411477057 tejrainn https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411479752

Since this was a proof of concept experiment for active protein delivery, future direction for this work would be incorporating this UCNP formulation for the delivery of other gene editing proteins, such as CRISPR-Cas9, which opens the doors allowing us to genetically engineer any organism with ease and efficiency. 

• For instance, this NP formulation might be advantageous in correcting mutations in subventricular zone (SVZ) cells.

Approach used to facilitate endosomal escape by the immobilized HCQ can certainly be incorporated into other delivery systems for RNA delivery, which may result in a more efficient and precise control of whatever it may be that is being administered, including a higher biosafety profile, increased cargo size capacity, and non specific infection of neurons.

UCNP formulation may lead to in vivo precise spatial control of gene expression, paired with optogenetics to measure the activity of the edited cells, resulting in improved gene editing. 

The developed UCNP formulation might help map specific neuronal circuits in vivo, as well as to image and track specific cell populations (Madisen et al., 2010).

Accumulation of UCNPs in the brain may be used as optogenetic actuators of transcranial NIR light to stimulate deep brain neurons (Chen et al., 2018). 

Moreover, this UCNP formulation brings up opportunities for in vivo precise spatial control of gene expression, paired with optogenetics to measure the activity of the edited cells, resulting in improved gene editing, which is the main focus on this paper.  

The authors hope that future studies are able to achieve higher cell recombination by improving the accumulation of Cre-UCNPs in the brain after intranasal instillation. 

]]> 2022-12-07 01:18:31 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411479752 tejrainn https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411482009

There seems to be little clinical significance of this paper and the authors’ findings

• Currently, this paper has only been cited once in a journal letter that emphasizes the advantage of NIR-mediated excitation in providing deep tissue photon penetration, making UCNPs appropriate for use in in vivo applications. 

This experimental method is extremely intricate in terms of the UCNP formulation and its actual impact on the field, proving that its simply too much effort for mediocre results. 

]]> 2022-12-07 01:20:37 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411482009 aledblundell https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411490421 Flow cytometry analysis shows experiments where only one element of the Cre-UCNP system is present leads to no recombination. All elements must be present for maximum recombination.]]> 2022-12-07 01:28:30 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411490421 aledblundell https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411494318 2022-12-07 01:32:23 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411494318 sheshataylor1 https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411497514 SVZ cells were cultured with Cre-UCNPs, washed, exposed/ not exposed to a NIR laser, and cultured for 4 more days to allow cells to undergo recombination ]]> 2022-12-07 01:35:23 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411497514 sheshataylor1 https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411499346 Cell recombination was evaluated by quantifying YFP-positive cells. Cre-UCNPs mediated gene editing had a higher efficiency than both soluble Cre (with HCQ) and AAV transfection with Cre (AAV5-Cre). ]]> 2022-12-07 01:37:20 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411499346 sheshataylor1 https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411503874 2022-12-07 01:42:11 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411503874 sheshataylor1 https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411506471 One of the main aims of this paper was to demonstrate gene editing in the ventral tegmental area to facilitate precise optogenetic control of reward and reinforcement as it has a lot of clinical significance. An AAV was injected into the VTA together with Cre-UCNPs. Following stereotaxic injection, the target site was irradiated by a NIR laser to induce the release of Cre from the NPs. After 4 weeks, animals were subjected to a CPP test (conditioned place preference), where the animal’s preference for one of two regions with contrasting visual and tactile cues in a cage was monitored for 3 days using an automated mouse tracking system. On day 4, whenever the mouse crossed into the non-preferred region of the cage, the VTA was activated with blue light; on the final test day, the animals were reintroduced into the cage to assess changes in preferences.

]]> 2022-12-07 01:44:58 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411506471 tejrainn https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411527933

Scientific future challenges lie with the dependency of the future directions of this work, such as improving the UCNP formulation, scaling up production of the UCNPs, higher cell recombination with intranasal instillation, and more.

• Also moving to human trials and having to adjust for differences or issues that may unintentionally arise 

Ethical future challenges would be related UCNPs involvement in gene editing and examining the ethical issues of gene editing itself such as germline editing, which means that changes will be passed down to future generations, and its overall side effects. 

• Also looking at who will be able to receive gene editing therapy (rich vs poor), etc.

]]> 2022-12-07 02:06:12 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411527933 aledblundell https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411547514 2022-12-07 02:24:56 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411547514 aledblundell https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411560626 AAVs delivered a sequence including inverted YFP and channelrhodopsin (ChR2) genes to isolated cortical neuron cells. In the presence of Cre recombinase, YFP and ChR2 are expressed.]]> 2022-12-07 02:38:42 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411560626 aledblundell https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411563034 Cortical neurons were isolated from mice, and cultured for 7 days. AAVs were delivered to install the reporter and ChR2 sequence (left of this post). After 3 days, Cre-UCNPs were added, and cells were washed after 4 hrs. Cells were then exposed to NIR light, and cultured for 3 days. Flow and ChR2 experiments were then conducted.]]> 2022-12-07 02:40:51 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411563034 aledblundell https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411566602 In contrast to fibroblast Cre-reporter lines, recombination only occured in ~26% of cells. Without NIR light exposure, recombination drops to ~1%. ]]> 2022-12-07 02:44:33 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411566602 sheshataylor1 https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411567413 To further demonstrate the potential of Cre-UCNPs, NPs were administered to the endogenous SVZ region of Rosa26-YFP mice by stereotaxic injection. One hour after administration some of the animals were stimulated with or without exterior NIR light. ]]> 2022-12-07 02:45:22 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411567413 sheshataylor1 https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411569061 One month following activation, the YFP signal was confirmed by immunostaining, and the fluorescence intensity and the percentage of YFP-positive cells were quantified in the region of interest. Results show successful gene editing of SVZ cells. ]]> 2022-12-07 02:46:55 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411569061 aledblundell https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411579608 Cell depolarization could be controlled by intensity of blue light.  After these results, In Vivo experiments were conducted.]]> 2022-12-07 02:57:21 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411579608 spencerabssy https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411613340 2022-12-07 03:33:25 UTC https://padlet.com/sheshataylor1/6ak3yox7sfjhc10p/wish/2411613340