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The regenerative potential of Muller glia (MG) is extraordinary in fish, poor in chick and terrible in mammals. In the chick model, MG readily reprogram into proliferating Muller glia-derived progenitor cells (MGPCs), but neuronal differentiation is very limited. The factors that suppress the neurogenic potential of MGPCs in the chick are slowly being revealed. Isoforms of Nuclear Factor I (NFI) are cell-intrinsic factors that limit neurogenic potential; these factors are required for the formation of MG in the developing mouse retina (Clark et al., 2019) and deletion of these factors reprograms MG into neuron-like cells in mature mouse retina (Hoang et al., 2020). Accordingly, we sought to characterize the patterns of expression NFIs in the developing, mature and damaged chick retina. In addition, we characterized patterns of expression of NFIs in the retinas of large mammals, pigs and monkeys. Using a combination of single cell RNA-sequencing (scRNA-seq) and immunolabeling we probed for patterns of expression. In embryonic chick, levels of NFIs are very low in early E5 (embryonic day 5) retinal progenitor cells (RPCs), up-regulated in E8 RPCs, further up-regulated in differentiating MG at E12 and E15. NFIs are maintained in mature resting MG, microglia and neurons. Levels of NFIs are reduced in activated MG in retinas treated with NMDA and/or insulin+FGF2, and further down-regulated in proliferating MGPCs. However, levels of NFIs in MGPCs were significantly higher than those seen in RPCs. Immunolabeling for NFIA and NFIB closely matched patterns of expression revealed in different types of retinal neurons and glia, consistent with findings from scRNA-seq. In addition, we find expression of NFIA and NFIB through progenitors in the circumferential marginal zone at the far periphery of the retina. We find similar patterns of expression for NFIs in scRNA-seq databases for pig and monkey retinas. Patterns of expression of NFIA and NFIB were validated with immunofluorescence in pig and monkey retinas wherein these factors were predominantly detected in MG and a few types of inner retinal neurons. In summary, NFIA and NFIB are prominently expressed in developing chick retina and by mature neurons and glia in the retinas of chicks, pigs and monkeys. Although levels of NFIs are decreased in chick, in MGPCs these levels remain higher than those seen in neurogenic RPCs. We propose that the neurogenic potential of MGPCs in the chick retina is suppressed by NFIs. This article is protected by copyright. All rights reserved.

Gene delivery to fertilized eggs is often the first step in creation of transgenic animals, CRISPR knock-out, or early developmental studies. The zona pellucida, a hardened glycoprotein matrix surrounding the mammalian fertilized eggs, often complicates gene delivery by forming a barrier against transfection reagents and viruses. High efficiency techniques to perforate or penetrate the zona allow for access and gene delivery to fertilized eggs. However, these techniques often rely on highly skilled technologists, are costly, and require specialized equipment for micromanipulation, laser perforation, or electroporation. Here, we report that adenoassociated viruses (AAVs) with serotypes 1 or DJ can efficiently diffuse across the zona to deliver genes without any manipulations to fertilized eggs. We observe lowered rates of embryo development after treatment of embryos with all AAV serotypes. However, we were able to reduce adverse effects on embryo development by exposing embryos to AAVs at later stages of in vitro development. AAVs have low immune response and do not incorporate into their host chromosomes to cause insertional mutations. Hence, AAVs can serve as a highly effective tool for transient delivery of genes to fertilized mammalian eggs.

In May of 2019, the adeno-associated virus (AAV)-based gene therapy onasemnogene abeparvovec-xioi (Zolgensma) became the second Food and Drug Administration (FDA)-approved gene therapy with designated use for infants diagnosed with spinal muscular atrophy (SMA). The decision came nearly 10 years after results of the first preclinical models were initially reported. While the journey was an arduous one, the approval was an indication of the remarkable success of the first in-human clinical trials. According to the traditional classification system of autosomal recessive SMA, of which there are multiple types with phenotypic variability, SMA type 1 is the most common and most severe and represents 45% of the SMA patient population. Children with SMA type 1 cannot lift their heads without assistance and do not live past their second birthday. With Zolgensma, the first treated children with SMA type 1 have reached 5 years of age and some of them achieved the ability to sit unassisted or even walk. In this article, we review the work that led to FDA approval with emphasis on the development of preclinical and clinical studies.

Human primary natural killer (NK) cells are being widely advanced for cancer immunotherapy. However, methods for gene editing of these cells have suffered low transduction rates, high cell death, and loss of transgene expression after expansion. Here, we developed a highly efficient method for site-specific gene insertion in NK cells using CRISPR (Cas9/RNP) and AAVs. We compared AAV vectors designed to mediate gene insertion by different DNA repair mechanisms, homology arm lengths, and virus concentrations. We then validated the method for site-directed gene insertion of CD33-specific CARs into primary human NK cells. CAR transduction was efficient, its expression remained stable after expansion, and it improved efficacy against AML targets.

Human peripheral blood natural killer (NK) cells have intense antitumor activity and have been used successfully in several clinical trials. Modifying NK cells with a chimeric antigen receptor (CAR) can improve their targeting and increase specificity. Recently, we described an efficient method for gene targeting in NK cells using Cas9/ribonucleoprotein (RNP) complexes. Here we combined this approach with single-stranded (ss) or self-complementary (sc) Adeno-associated virus (AAV)-mediated gene delivery for gene insertion into a safe-harbor locus using a wide variety of homology arms for homology repair (HR) and non-homologous directed CRISPR-assisted insertion tagging (CRISPaint) approaches. For proof-of-concept, we generated mCherry-expressing primary NK cells and determined that sc vectors with 300bp homology arms had optimal transduction efficiency. Then, we generated CD33-targeting CAR NK cells with differing transmembrane and signaling domains (CD4/4-1BB+CD3ζ and NKG2D/2B4+CD3ζ) and expanded them on CSTX002 feeder cells. Expansion kinetics were unaltered and the expanded NK cells maintained high CAR expression (mean 68% CAR+). The CD33-CAR-NK cells showed increased activation markers and enhanced antileukemic activity with improved killing kinetics against CD33-positive acute myeloid leukemia (AML) cell lines and primary samples. Using targeted sequencing we demonstrated the accuracy of CAR gene insertion in human primary NK cells genome. Site-directed insertion using RNP and scAAV6 is an efficient method for stable genetic transfer into primary NK cells that has broad potential for fundamental discovery and therapeutic applications.

NEDAMSS is a newly discovered neurodevelopmental disorder with regression, abnormal movements, loss of speech and seizures. The disease is caused by heterozygous mutations in the Interferon Regulatory Factor 2 Binding Protein-Like (IRF2BPL) gene. IRF2BPL is a ubiquitously expressed intron-less transcriptional regulator. The function of the protein is currently unknown, but the observed disease phenotype indicates a role in development and maintenance of the nervous system. To study this disease, we used patient skin-derived fibroblasts and reprogrammed them into induced neuronal progenitor cells (iNPCs). The iNPCs were then differentiated into astrocytes. Moreover, we also used a direct conversion method to generate neurons directly from the skin fibroblasts. Using these methods and performing molecular and functional analyses of the different cell types, we identified a novel disease mechanism not previously described. While IRF2BPL is localized in the nucleus in healthy individuals, we found that several patient cell lines displayed mis-localization in the form of aggregates in the cytoplasm. Moreover, the patient astrocytes also showed abnormal mitochondrial ATP-linked respiration levels and caused neuronal death in co-culture assays. Based on these observations, we tested treatment with CuATSM, a small molecule drug currently in clinical trials for ALS. We found that CuATSM successfully rescued neuronal survival and reduced ATP-linked respiration to normal levels in NEDAMSS astrocytes. We also established multiple AAV based gene therapy tools to modulate the expression of the protein and restore its normal function in vitro and potentially in vivo. In summary, we identified a novel disease mechanism involved in NEDAMSS disease and found promising therapeutic strategies that might be able to slow or halt the progression of the disease.

Human peripheral blood natural killer (NK) cells have intense antitumor activity and have been used successfully in several clinical trials. Modifying NK cells with a chimeric antigen receptor (CAR) can improve their targeting and increase specificity, but genetic modification of primary human NK cells has been problematic compared to the ease in which primary human T cells are transduced. Despite some progress made in using lentiviral and retroviral vectors, the efficiency of viral transduction in NK cells remains relatively low. Additionally, using these viral vectors results in random integration events which can potentially affect the safety or efficacy of transduced immune cells. Recently, we described an efficient method for gene targeting in primary human NK cells using Cas9/ribonucleoprotein (RNP) complexes. Here we combined that approach with Adeno-associated virus (AAV)-mediated gene delivery for gene insertion into a safe-harbor locus, and optimized transduction efficiency using various AAV pseudotypes, single-stranded (ss) or self-complementary (sc) with a wide variety of homology arms, and homology repair (HR) andor non-homologous directed CRISPR-assisted insertion tagging (CRISPaint) approaches. For initial optimization we generated mCherry-expressing primary NK cells and determined that sc vectors with 300bp homology arms for HR resulted in optimal transduction efficiency. To validate the process for generating large numbers of CAR-NK cells needed for clinical application, we generated CD33-targeting CAR NK cells with differing transmembrane and signaling domains (CD4/4-1BB+CD3ζ and NKG2D/2B4+CD3ζ) and expanded them on CSTX002 feeder cells. Expansion kinetics were unaltered by the transgene, and the NK cells maintained high CAR expression (mean 68% CAR+) during expansion. The expanded CD33-CAR-NK cells showed enhanced activation and antileukemic activity with improved killing kinetics against CD33-positive acute myeloid leukemia (AML). Using targeted sequencing we demonstrated the accuracy of CAR gene insertion in human primary NK cells genome and low off-target insertion. Site-directed insertion using RNP and scAAV6 is an efficient method for stable genetic transfer into primary NK cells that has broad potential for fundamental discovery and therapeutic applications.

Noise-induced hearing loss (NIHL) is one of the most prevalent disabilities for which effective therapeutic treatment is currently lacking. Auditory injury caused by excessive or constant noise exposure damages the sensory elements of the ear, leading to metabolic or mechanical damage to hair cells and subsequent degeneration of spiral ganglion neurons (SGNs). To improve hearing in the NIHL patient population, maintenance and regeneration of inner hair cell-SGN synapses is critical. Although effective compounds for the treatment of NIHL have been suggested, challenges in delivery of the compounds to the inner hair cells (IHCs) hinder translation of these therapies to a clinical setting. Current routes of administration are ineffective in reaching IHCs or are invasive and may damage the cochlea. In this study, we examined less invasive delivery routes, via intrathecal (cerebrospinal fluid delivery) or intravenous injection, to administer adeno-associated virus serotype 9 (AAV9) expressing green fluorescent protein (GFP) to target IHCs and SGNs. To induce auditory injury, mice were exposed to 100 dB sound pressure level (SPL) octave band noise for 2 hours. Mice were then injected at various time points post noise injury to determine if noise exposure influenced targeting efficiency. Three weeks post injection, cochleae were processed and analyzed for GFP expression using immunofluorescence. Our results demonstrate that intravenous delivery was not successful in targeting of IHCs or SGNs at any time point measured. However, intrathecally injected AAV9 was highly effective in targeting cells in both healthy and noise-damaged cochleae at multiple time points post-noise exposure. We conclude that gene therapy techniques can be utilized to efficiently deliver therapeutic transgenes to the cochlea using cerebrospinal fluid as a delivery route. These findings determine a novel route of delivery and open a new avenue of therapeutic treatment for auditory injury and cochlear disorders.

Batten Disease is a fatal, lysosomal storage disorder characterized by cognitive and motor deficits, vision impairments, and seizures. Loss of vision is a hallmark of 10 of the 13 Batten Disease subtypes. Our group has pioneered AAV9 gene therapy treatments achieving widespread transduction of the brain and spinal cord. Two clinical trials are currently ongoing at Nationwide Children’s Hospital delivering this vector via cerebrospinal fluid (CSF) to the brain and spinal cord for treatment of the lethal neurodegenerative aspects of Batten Disease. However, AAV9 transduction of the retina after CSF delivery is limited and there is a critical need for a solution that prevents vision loss and further improves quality of life for Batten Disease patients. Similar to most genetic ocular diseases, photoreceptor degeneration is the most commonly cited pathology in patients. However, recent studies suggest that in some subtypes of Batten Disease, expression must be rescued also within the deeper layers of the retina that are difficult to reach with therapeutic vectors (inner nuclear layer, INL). We performed single cell RNA sequencing of mice and non-human primate retinas in collaboration with Dr. Fischer (OSU) and concluded that Batten Disease vision specific gene therapy needs to target a wide range of cells, including the INL, which is a major challenge for translation of a vision specific therapy to the clinic. We have recently discovered that administration of neuraminidase (NA), a sialidase enzyme, prior to or in combination with AAV9.GFP, drastically increases transduction throughout the murine C57Bl/6 retina including the INL and all the way through to the photoreceptor layer. Our preliminary data indicates GFP expression in almost all, if not all, retinal cell types using this method. Importantly, we have confirmed successful targeting of INL bipolar cells, a notoriously difficult cell-type to transduce, and up to 40% increase in Muller glia transduction. Preliminary histological examination indicates no damage or alterations in retinal integrity. While additional testing in large animal models is required and scheduled to occur in a WT pig model in Autumn 2020, this remarkable discovery suggests that it may now be possible to target every cell-type of the retina with a single AAV vector. This is especially important in regard to treatment of Batten Disease but has many additional implications. Current retinal therapies that require a more invasive subretinal delivery to photoreceptors could now opt for the safer intravitreal delivery strategy. In addition, this strategy would be highly useful in the field of optogenetics, where investigators continue to struggle to find an efficient way to express light-sensitive opsins in cells of the INL to restore sight in individuals that have already lost their photoreceptors.

Human peripheral blood natural killer (NK) cells have intense antitumor activity and have been used successfully in several clinical trials. Modifying NK cells with a chimeric antigen receptor (CAR) can improve their targeting and increase specificity. Recently, we described an efficient method for gene targeting in NK cells using Cas9/ribonucleoprotein complexes (PMID: 29985369 and 32603414). Here we combined this approach with single-stranded (ss) or self-complementary (sc) Adeno-associated virus (AAV)-mediated gene delivery for gene insertion into a safe-harbor locus using a wide variety of homology arms for homology repair (HR) and non-homologous directed CRISPR-assisted insertion tagging (CRISPaint) approaches. We demonstrated that expansion of NK cells on feeder cells (CSTX002) expressing membrane-bound IL21 increases expression of HDR-related genes and provides optimum biological condition for targeted gene insertion. For proof-of-concept, we successfully generated stable mCherry-expressing primary NK cells (up to 89% mCherry+) and determined that sc vectors with 300bp homology arms were optimal. Then, we generated CD33-targeting CAR NK cells with differing transmembrane and signaling domains (CD4/4-1BB+CD3ζ and NKG2D/2B4+CD3ζ), which continued to show robust expansion on CSTX002 and stably maintained their CAR expression. This resulted in CAR-NK-cells of high number and purity (mean 68% CAR+) that demonstrated enhanced antileukemic activity against acute myeloid leukemia (AML) cell lines. This efficient method for site-directed insertion of genetic materials into primary NK cells has broad potential for fundamental discovery and therapeutic applications. Keywords: CRISPR, NK, Cas9/RNP, AAV6, CRISPaint, HR, CD33CAR-NK Figure Disclosures Naeimi Kararoudi: Kiadis Pharma Netherlans B.V: Patents & Royalties. Lee:Kiadis Pharma Netherlands B.V: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.