Biology subjects

Gamm, D. M.

Publications and source records attributed to Gamm, D. M..

8 recordsLinked to original sources

bioRxiv2026

CTCF regulates wild-type and recombinant AAV gene expression by shaping viral chromatin

Adeno-Associated Viruses (AAVs) are powerful platforms for delivering therapeutic transgenes via recombinant AAV (rAAV) vectors. However, a limited understanding of the regulation of AAV gene expression has narrowed the ability to efficiently express therapeutic transgenes from rAAV vectors. Since rAAVs retain only the wtAAV inverted terminal repeats (ITR), we hypothesized that regulatory elements outside the ITR that govern wild-type AAV (wtAAV) gene expression can be used to modify rAAV genomes to enhance vector performance. Through in silico analysis, biochemical pulldowns, and high-throughput sequencing, we have identified that the host architectural protein CCCTC-binding Factor (CTCF) associates with the wtAAV type 2 (wtAAV2) genome but is absent from rAAV vectors. Global knockdown and site-specific deletion revealed that the CTCF binding element (CBE) on the wtAAV2 genome, located upstream of the viral P5 promoter, regulates expression of the viral Rep68/78 genes. We have re-engineered new rAAV vectors expressing a GFP reporter transgene to contain the wtAAV2-CBE upstream of the vector promoter. Our results show that CTCF binding dramatically increased rAAV transduction efficiency and GFP expression by up to four-fold across multiple cell types. This enhancement was independent of the AAV capsid serotype used for packaging rAAV vectors. CUT&RUN analysis revealed that this CBE was necessary and sufficient to regulate the chromatin landscape of wtAAV2 and rAAV2. Finally, we observed that CTCF-mediated chromatin remodeling of rAAV2 led to increased production of nascent RNA transcripts from the vector genome. Based on our findings, we propose that CTCF supports wtAAV2/rAAV gene expression by shaping the local chromatin landscape. SIMPLE ABSTRACTRecombinant Adeno-Associated Viruses (rAAV) gene therapy vectors have been engineered from wild-type AAV (wtAAV) by inserting the viral telomeres (that serve as replication and packaging signals) on either side of therapeutic transgenes. However, efficient expression of transgenes using current rAAV technologies require high doses, which can lead to sporadic toxic side effects. We hypothesized that uncharacterized regulatory elements in the wtAAV2 genome drive efficient viral gene expression and are absent from the current generation of rAAV vectors. Using in-silico analysis combined with biochemical pulldowns, high-throughput sequencing, and mutant viral systems, we have identified a novel cis-acting element bound by the cellular architectural protein CCCTC-binding Factor (CTCF). This CTCF binding element is necessary for wtAAV2 gene expression and is sufficient to enhance the rAAV vectors ability to express reporter transgenes. This CTCF-binding element regulates the chromatin landscape of the virus and its vectors. Our discovery that adding the 19-bp AAV2 CTCF-binding element enhances transgene expression without affecting vector production efficiency presents a promising new rAAV gene therapy platform that is likely to reduce clinical doses and minimize toxicity in therapeutic applications.

microbiology↗

bioRxiv2025

Prevenying vision loss in a mouse model of Leber Congenital Amaurosis by engineered tRNA

Premature termination codons (PTCs) are associated with rare genetic disorders. Inducing targeted read-through of these nonsense mutations presents a potential therapeutic strategy for modifying disease outcomes. We previously reported that one such PTC, W53X, in the KCNJ13 gene causes blindness and Leber congenital amaurosis type-16 (LCA-16) due to loss of function of the inwardly rectifying potassium channel 7.1 (Kir7.1). Here, we present the proof of concept of a therapeutic approach based on anticodon-engineered transfer RNA (ACE-tRNA). The ACE-tRNA encodes the amino acid tryptophan (Trp) and suppresses the W53X PTC, restoring full-length protein expression. We used helper-dependent adenovirus (HDAd) to deliver the ACE-tRNATrp.UAG (tRNATrp.UAG) and rescue Kir7.1 function and physiology in patient-specific human induced pluripotent stem cell-derived retinal pigment epithelium (hiPSC-RPE) cells. Furthermore, in a W53X mouse model of LCA16, HDAd delivery of tRNATrp.UAG resulted in durable restoration of vision as measured by retinography. This study provides the first example of the therapeutic application of ACE-tRNA for treating an inherited form of blindness.

physiology↗

bioRxiv2025

Genome-Wide CRISPR Screening Identifies Cellular Factors Controlling Nonviral Genome Editing Efficiency

After administering genome editors, their efficiency is limited by a multi-step process involving cellular uptake, trafficking, and nuclear import of the vector and its payload. These processes vary widely across cell types and differ depending on the nature and structure of the vector, whether it is a lipid nanoparticle or a different synthetic material. We developed a novel genome-wide CRISPR screening strategy to better understand these limitations within human cells to identify genes modulating cellular uptake, payload delivery, and gene editing efficiency. Our screen interrogates the cellular processes controlling genome editing by Cas-based nuclease and base editing strategies in human cells. We designed a genome-wide screen targeting 19,114 genes in HEK293 cells, and we identified six genes whose knockout increased nonviral editing efficiency in human cells by up to five-fold. Further validation through arrayed knockouts of the top hits from our screen boosted the editing efficiency from 5% to 50% when Cas9 was delivered via lipid-based nanoparticles. By designing the guides to target the screen library cassette, we could accurately track the library sgRNA identity and the editing outcome on the same amplicon via short-read sequencing, enabling the identification of rare outcomes via computationally sorting edited from unedited cells within a heterogenous pool of >200M cells. In patient-derived human retinal pigment epithelium cells derived from pluripotent stem cells, BET1L, GJB2, and MS4A13 gene knockouts increased targeted genome editing by over five-fold. We anticipate that this high-throughput screening approach will facilitate the systematic engineering of novel nonviral genome editing delivery methods, where the identified novel gene hits can be further used to increase editing efficiency for other therapeutically relevant cell types.

genomics↗

bioRxiv2024

Evaluation of subretinally delivered Cas9 ribonucleoproteins in murine and porcine animal models highlights key considerations for therapeutic translation of genetic medicines

Genetic medicines, including CRISPR/Cas technologies, extend tremendous promise for addressing unmet medical need in inherited retinal disorders and other indications; however, there remain challenges for the development of therapeutics. Herein, we evaluate genome editing by engineered Cas9 ribonucleoproteins (eRNP) in vivo via subretinal administration using mouse and pig animal models. Subretinal administration of adenine base editor and double strand break-inducing Cas9 nuclease eRNPs mediate genome editing in both species. Editing occurs in retinal pigmented epithelium (RPE) and photoreceptor cells, with favorable tolerability in both species. Using transgenic reporter strains, we determine that editing primarily occurs close to the site of administration, within the bleb region associated with subretinal injection. Our results show that subretinal administration of eRNPs in mice mediates base editing of up to 12% of the total neural retina, with an average rate of 7% observed at the highest dose tested. In contrast, a substantially lower editing efficiency was observed in minipigs; even with direct quantification of only the treated region, a maximum base editing rate of 1.5%, with an average rate of <1%, was observed. Our data highlight the importance of species consideration in translational studies for genetic medicines targeting the eye and provide an example of a lack of translation between small and larger animal models in the context of subretinal administration of Cas9 eRNPs.

pharmacology and toxicology↗

bioRxiv2021

Human pluripotent stem cell-derived photoreceptors switch from cell autonomous axon extension to non-cell autonomous process pulling during synaptic marker redistribution.

Photoreceptors (PRs) are the primary visual sensory cells, and their loss leads to blindness that is currently incurable. Cell replacement therapy holds promise as a therapeutic approach to restore vision to those who have lost PRs through damage or disease. While PR transplant research is ongoing in animal models, success is hindered by our limited understanding of PR axon growth during development and regeneration. Using a human pluripotent stem cell (hPSC) reporter line that labels PRs (WA09 CRX+/tdTomato), we generated retinal organoids in order to study mechanisms of PR process extension. We found that the earliest born PRs exhibit autonomous axon extension from dynamic terminals that appear similar to projection neuron growth cones. However, as hPSC-derived PRs age from 40 to 80 days of differentiation, they lose dynamic terminals in 2D plated cultures and within 3D retinal organoids, which does not correlate with cell birth date. Using a rod-specific hPSC reporter line (WA09 NRL+/eGFP), we further determined that rod PRs never form motile growth cones. Interestingly, PRs without motile terminals are still capable of extending axons, but neurites are generated from process stretching via their attachment to motile non-PR cells, which underlies the observed differences in PR neurite lengths on different substrata. While immobile PR terminals express actin, it is less polymerized and less organized than actin present in motile terminals. However, immobile PRs do localize synaptic proteins to their terminals, suggesting a normal developmental progression. These findings help inform the development of PR transplant therapies to treat blinding diseases and provide a platform to test treatments that restore autonomous PR axon extension. Significance StatementLoss of photoreceptors (PRs) in the retina through damage or disease causes irreversible vision loss and blindness. One treatment approach is to replace lost cells with transplanted human stem cell-derived PRs, but this requires PR axons to integrate into the host retina to restore the required neural connections. For this strategy to succeed, we need to understand how PRs extend processes to their targets during development in situ, and whether dissociated human stem cell (hPSC)-derived PRs behave in a similar fashion. In this paper, we show that hPSC-PRs have only a short window during which they are capable of autonomous axon extension, which has implications for PR transplant efforts and for our basic understanding of human retinal development.

neuroscience↗

bioRxiv2019

Human iPSC modeling elucidates mutation-specific responses to gene therapy in a genotypically diverse dominant maculopathy

Dominantly inherited disorders are not typically considered therapeutic candidates for gene augmentation. Here, we utilized patient-specific induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE) to test the potential of gene augmentation to treat Best disease, a dominant macular dystrophy caused by over 200 missense mutations in BEST1. Gene augmentation in iPSC-RPE fully restored BEST1 calcium-activated chloride channel activity and improved rhodopsin degradation in iPSC-RPE models of recessive bestrophinopathy and dominant Best disease caused by two different ion binding domain mutations. A dominant Best disease iPSC-RPE model that did not respond to gene augmentation showed normalization of BEST1 channel activity following CRISPR-Cas9 editing of the mutant allele. We then tested gene editing in all three dominant Best disease iPSC-RPE models, which produced premature stop codons exclusively within the mutant BEST1 alleles. Single-cell profiling demonstrated no adverse perturbation of RPE transcriptional programs in any model, although off-target analysis detected a silent genomic alteration in one model. These results suggest that gene augmentation is a viable first-line approach for some dominant Best disease patients and that non-responders are candidates for alternate approaches such as genome editing. However, testing genome editing strategies for on-target efficiency and off-target events using patient-matched iPSC-RPE model systems is warranted. In summary, personalized iPSC-RPE models can be used to select among a growing list of gene therapy options to maximize safety and efficacy while minimizing time and cost. Similar scenarios likely exist for other genotypically diverse channelopathies, expanding the therapeutic landscape for affected patients. SignificanceDominantly inherited disorders pose distinct challenges for gene therapies, particularly in the face of extreme mutational diversity. We tested whether a broad gene replacement strategy could reverse the cellular phenotype of Best disease, a dominant blinding condition that targets retinal pigment epithelium (RPE). Using RPE generated from patient-specific induced pluripotent stem cells (iPSCs), we show that gene replacement functionally overcomes some, but not all, of the tested mutations. In comparison, all dominant Best disease models tested were phenotypically corrected after mutation-specific genome editing, although one off-target genomic alteration was discovered. Our results support a two-tiered approach to gene therapy for Best disease, guided by safety and efficacy testing in iPSC-RPE models to maximize personal and public health value.

genetics↗

bioRxiv2019

A combined RNA-seq and whole genome sequencing approach for identification of non-coding pathogenic variants in single families.

Inherited retinal degenerations (IRDs) are at the focus of current genetic therapeutic advancements. For a genetic treatment such as gene therapy to be successful an accurate genetic diagnostic is required. Genetic diagnostics relies on the assessment of the probability that a given DNA variant is pathogenic. Non-coding variants present a unique challenge for such assessments as compared to coding variants. For one, non-coding variants are present at much higher number in the genome than coding variants. In addition, our understanding of the rules that govern the non-coding regions of the genome is less complete than our understanding of the coding regions. Methods that allow for both the identification of candidate non-coding pathogenic variants and their functional validation may help overcome these caveats allowing for a greater number of patients to benefit from advancements in genetic therapeutics. We present here an unbiased approach combining whole genome sequencing (WGS) with patient induced pluripotent stem cell (iPSC) derived retinal organoids (ROs) transcriptome analysis. With this approach we identified and functionally validated a novel pathogenic non-coding variant in a small family with a previously unresolved genetic diagnosis.

genetics↗

bioRxiv2018

Gene augmentation and read-through rescue channelopathy in an iPSC-RPE model of congenital blindness

PurposeMutations in the KCNJ13 gene are known to cause Lebers Congenital Amaurosis (LCA16), an inherited pediatric blindness. KCNJ13 gene encodes the Kir7.1 subunit protein which acts as a tetrameric inwardly rectifying potassium ion channel in the retinal pigment epithelium to maintain ionic homeostasis thereby allowing photoreceptors to encode visual information. We sought to determine if genetic approaches might be effective in treating blindness due to mutations in KCNJ13.\n\nMethodsWe developed patient-derived hiPSC-RPE carrying an autosomal recessive nonsense mutation in the KCNJ13 gene (c.158G>A, p.Trp53*). We performed biochemical and electrophysiology assays of Kir7.1 function. Both small molecule read-through drug and gene-therapy approaches were tested using this disease-in-a-dish approach.\n\nResultsWe found that the LCA16 hiPSC-RPE had normal morphology but did not express a functional Kir7.1 channel and was unable to demonstrate normal physiology. Following read-through drug treatment, the LCA16 hiPSC cells were hyperpolarized by 30 mV and Kir7.1 current was restored. Similarly, loss-of-function of Kir7.1 channel was circumvented by lentiviral gene delivery to the hiPSC-RPE cells. In either approach, Kir7.1 protein was expressing normally with restoration of membrane potential and Kir7.1 current.\n\nConclusionLoss-of-function mutation in Kir7.1 is a cause of LCA. Using either read-through therapy or gene augmentation, we rescued Kir7.1 channel function in patient-derived iPSC-RPE cells via a precision medicine approach.

genetics↗