Gene Therapy

Gene Therapy Publications

Chan YK, Wang SK, Chu CJ, Copland DA, Letizia AJ, Costa Verdera H, Chiang JJ, Sethi M, Wang MK, Neidermyer WJ, Chan Y, Lim ET, Graveline AR, Sanchez M, Boyd RF, Vihtelic TS, Inciong RGCO, Slain JM, Alphonse PJ, Xue Y, Robinson-McCarthy LR, Tam JM, Jabbar MH, Sahu B, Adeniran JF, Muhuri M, Tai PWL, Xie J, Krause TB, Vernet A, Pezone M, Xiao R, Liu T, Wang W, Kaplan HJ, Gao G, Dick AD, Mingozzi F, McCall MA, Cepko CL, Church GM. Engineering adeno-associated viral vectors to evade innate immune and inflammatory responses. Sci Transl Med 2021;13(580)Abstract
Nucleic acids are used in many therapeutic modalities, including gene therapy, but their ability to trigger host immune responses in vivo can lead to decreased safety and efficacy. In the case of adeno-associated viral (AAV) vectors, studies have shown that the genome of the vector activates Toll-like receptor 9 (TLR9), a pattern recognition receptor that senses foreign DNA. Here, we engineered AAV vectors to be intrinsically less immunogenic by incorporating short DNA oligonucleotides that antagonize TLR9 activation directly into the vector genome. The engineered vectors elicited markedly reduced innate immune and T cell responses and enhanced gene expression in clinically relevant mouse and pig models across different tissues, including liver, muscle, and retina. Subretinal administration of higher-dose AAV in pigs resulted in photoreceptor pathology with microglia and T cell infiltration. These adverse findings were avoided in the contralateral eyes of the same animals that were injected with the engineered vectors. However, intravitreal injection of higher-dose AAV in macaques, a more immunogenic route of administration, showed that the engineered vector delayed but did not prevent clinical uveitis, suggesting that other immune factors in addition to TLR9 may contribute to intraocular inflammation in this model. Our results demonstrate that linking specific immunomodulatory noncoding sequences to much longer therapeutic nucleic acids can "cloak" the vector from inducing unwanted immune responses in multiple, but not all, models. This "coupled immunomodulation" strategy may widen the therapeutic window for AAV therapies as well as other DNA-based gene transfer methods.
Ballios BG, Pierce EA, Huckfeldt RM. Gene editing technology: Towards precision medicine in inherited retinal diseases. Semin Ophthalmol 2021;:1-9.Abstract
To review preclinical and clinical advances in gene therapy, with a focus on gene editing technologies, and application to inherited retinal disease. A narrative overview of the literature, summarizing the state-of-the-art in clinical gene therapy for inherited retinal disease, as well as the science and application of new gene editing technology. The last three years has seen the first FDA approval of an in vivo gene replacement therapy for a hereditary blinding eye disease and, recently, the first clinical application of an in vivo gene editing technique. Limitations and challenges in this evolving field are highlighted, as well as new technologies developed to address the multitude of molecular mechanisms of disease. Genetic therapy for the treatment of inherited retinal disease is a rapidly expanding area of ophthalmology. New technologies have revolutionized the field of genome engineering and rekindled an interest in precision medicines for these conditions.
Yang S, Kam WR, Liu Y, Ding J, Li Y, Sullivan DA. Comparative influence of differentiation and proliferation on gene expression in human meibomian gland epithelial cells. Exp Eye Res 2021;205:108452.Abstract
We recently discovered that by changing environmental signals, differentiated immortalized human meibomian gland epithelial cells (IHMGECs) de-differentiate into proliferating cells. We also discovered that following exposure to appropriate stimuli, these proliferative cells re-differentiate into differentiated IHMGECs. We hypothesize that this plasticity of differentiated and proliferative IHMGECs is paralleled by very significant alterations in cellular gene expression. To begin to test this hypothesis, we compared the gene expression patterns of IHMGECs during differentiation and proliferation. IHMGECs were cultured for four days in either differentiating or proliferating media. After four days of culture, cells were processed for the analysis of gene expression by using Illumina BeadChips and bioinformatic software. Our study identified significant differences in the expression of more than 9200 genes in differentiated and proliferative IHMGECs. Differentiation was associated with significant increases in the expression of specific genes (e.g. S100 calcium binding protein P; 7,194,386-fold upregulation) and numerous ontologies (e.g. 83 biological process [bp] ontologies with ≥100 genes were upregulated), such as those related to development, transport and lysosomes. Proliferation also led to a significant rise in specific gene expressions (e.g. cathelicidin antimicrobial peptide; 859,100-fold upregulation) and many ontologies (115 biological process [bp] ontologies with ≥100 genes were upregulated), with most of the highly significant ontologies related to cell cycle (z scores > 13.9). Our findings demonstrate that gene expression in differentiated and proliferative IHMGECs is extremely different. These results may have significant implications for the regeneration of HMGECs and the reversal of MG dropout in MG dysfunction.
Hildebrandt C, Fulton A, Rodan LH. Homozygous deletion of 21q22.2 in a patient with hypotonia, developmental delay, cortical visual impairment, and retinopathy. Am J Med Genet A 2021;185(2):555-560.Abstract
21q22 contains several dosage sensitive genes that are important in neurocognitive development. Determining impacts of gene dosage alterations in this region can be useful in establishing contributions of these genes to human development and disease. We describe a 15-month-old girl with a 1,140 kb homozygous deletion in the Down Syndrome Critical Region at 21q22.2 including 4 genes; B3GALT5, IGSF5, PCP4, DSCAM, and a microRNA (MIR4760). Clinical singleton genome sequencing did not report any candidate gene variants for the patient's phenotype. She presented with hypotonia, global developmental delay, cortical visual impairment, and mild facial dysmorphism. Ophthalmological exam was suggestive of retinopathy. We propose that the absence of DSCAM and PCP4 may contribute to the patient's neurological and retinal phenotype, while the role of absent B3GALT5 and IGSF5 in her presentation remain unclear at this time.
Jacob A, Brun L, Gil PJ, Ménard L, Bouzelha M, Broucque F, Roblin A, Vandenberghe LH, Adjali O, Robin C, François A, Blouin V, Penaud-Budloo M, Ayuso E. Homologous Recombination Offers Advantages Over Transposition-Based Systems to Generate Recombinant Baculovirus for Adeno-Associated Viral Vector Production. Biotechnol J 2020;:e2000014.Abstract
Viral vectors have a great potential for gene delivery, but manufacturing at a pharmaceutical scale is a big challenge for the industry. The baculovirus-insect cell system is one of the most scalable platforms to produce clinical-grade recombinant Adeno-Associated Virus (rAAV) vectors. The standard procedure to generate recombinant baculovirus is based on Tn7 transposition which is time-consuming and still suffers technical constraints. Moreover, werecently showed that baculoviral sequences adjacent to the AAV ITRs are preferentially encapsidated into the rAAV vector particles. This observation raised concerns about safety for clinical applications due to the presence of bacterial and antibiotic resistance coding sequences with a Tn7-mediated system for the construction of baculoviruses reagents. Here, weinvestigated a faster and safer method based on homologous recombination (HR). First, weconfirmed the functionality of the inserted cassette and the absence of undesirable genes into HR-derived baculoviral genomes. Strikingly, wefound that the exogenous cassette showed increased stability over passages when using the HR system. Finally, wetested these materials to produce rAAV vectors. The baculoviruses originated from both systems lead to high rAAV vector genome yields, with the advantage of the HR system being exempted from undesirable bacterial genes which provides an additional level of safety for the manufacturing of rAAV vectors. Overall, this study highlights the importance of the upstream process and starting biologic materials to generate safer rAAV biotherapeutic products. This article is protected by copyright. All rights reserved.
Hu Z, Cano I, Saez-Torres KL, LeBlanc ME, Saint-Geniez M, Ng Y-S, Argüeso P, D'Amore PA. Elements of the Endomucin Extracellular Domain Essential for VEGF-Induced VEGFR2 Activity. Cells 2020;9(6)Abstract
Endomucin (EMCN) is the type I transmembrane glycoprotein, mucin-like component of the endothelial cell glycocalyx. We have previously shown that EMCN is necessary for vascular endothelial growth factor (VEGF)-induced VEGF receptor 2 (VEGFR2) internalization and downstream signaling. To explore the structural components of EMCN that are necessary for its function and the molecular mechanism of EMCN in VEGF-induced endothelial functions, we generated a series of mouse EMCN truncation mutants and examined their ability to rescue VEGF-induced endothelial functions in human primary endothelial cells (EC) in which endogenous EMCN had been knocked down using siRNA. Expression of the mouse full-length EMCN (FL EMCN) and the extracellular domain truncation mutants ∆21-81 EMCN and ∆21-121 EMCN, but not the shortest mutant ∆21-161 EMCN, successfully rescued the VEGF-induced EC migration, tube formation, and proliferation. ∆21-161 EMCN failed to interact with VEGFR2 and did not facilitate VEGFR2 internalization. Deletion of COSMC (C1GalT1C1) revealed that the abundant mucin-type -glycans were not required for its VEGFR2-related functions. Mutation of the two -glycosylation sites on ∆21-121 EMCN abolished its interaction with VEGFR2 and its function in VEGFR2 internalization. These results reveal ∆21-121 EMCN as the minimal extracellular domain sufficient for VEGFR2-mediated endothelial function and demonstrate an important role for -glycosylation in VEGFR2 interaction, internalization, and angiogenic activity.
Amamoto R, Zuccaro E, Curry NC, Khurana S, Chen H-H, Cepko CL, Arlotta P. FIN-Seq: transcriptional profiling of specific cell types from frozen archived tissue of the human central nervous system. Nucleic Acids Res 2020;48(1):e4.Abstract
Thousands of frozen, archived tissue samples from the human central nervous system (CNS) are currently available in brain banks. As recent developments in RNA sequencing technologies are beginning to elucidate the cellular diversity present within the human CNS, it is becoming clear that an understanding of this diversity would greatly benefit from deeper transcriptional analyses. Single cell and single nucleus RNA profiling provide one avenue to decipher this heterogeneity. An alternative, complementary approach is to profile isolated, pre-defined cell types and use methods that can be applied to many archived human tissue samples that have been stored long-term. Here, we developed FIN-Seq (Frozen Immunolabeled Nuclei Sequencing), a method that accomplishes these goals. FIN-Seq uses immunohistochemical isolation of nuclei of specific cell types from frozen human tissue, followed by bulk RNA-Sequencing. We applied this method to frozen postmortem samples of human cerebral cortex and retina and were able to identify transcripts, including low abundance transcripts, in specific cell types.
Berry JL, Polski A, Cavenee WK, Dryja TP, Murphree LA, Gallie BL. The Story: Characterization and Cloning of the First Tumor Suppressor Gene. Genes (Basel) 2019;10(11)Abstract
The gene is the first described human tumor suppressor gene and plays an integral role in the development of retinoblastoma, a pediatric malignancy of the eye. Since its discovery, the stepwise characterization and cloning of have laid the foundation for numerous advances in the understanding of tumor suppressor genes, retinoblastoma tumorigenesis, and inheritance. Knowledge of led to a paradigm shift in the field of cancer genetics, including widespread acceptance of the concept of tumor suppressor genes, and has provided crucial diagnostic and prognostic information through genetic testing for patients affected by retinoblastoma. This article reviews the long history of gene research, characterization, and cloning, and also discusses recent advances in retinoblastoma genetics that have grown out of this foundational work.
Arboleda-Velasquez JF, Lopera F, O'Hare M, Delgado-Tirado S, Marino C, Chmielewska N, Saez-Torres KL, Amarnani D, Schultz AP, Sperling RA, Leyton-Cifuentes D, Chen K, Baena A, Aguillon D, Rios-Romenets S, Giraldo M, Guzmán-Vélez E, Norton DJ, Pardilla-Delgado E, Artola A, Sanchez JS, Acosta-Uribe J, Lalli M, Kosik KS, Huentelman MJ, Zetterberg H, Blennow K, Reiman RA, Luo J, Chen Y, Thiyyagura P, Su Y, Jun GR, Naymik M, Gai X, Bootwalla M, Ji J, Shen L, Miller JB, Kim LA, Tariot PN, Johnson KA, Reiman EM, Quiroz YT. Resistance to autosomal dominant Alzheimer's disease in an APOE3 Christchurch homozygote: a case report. Nat Med 2019;25(11):1680-1683.Abstract
We identified a PSEN1 (presenilin 1) mutation carrier from the world's largest autosomal dominant Alzheimer's disease kindred, who did not develop mild cognitive impairment until her seventies, three decades after the expected age of clinical onset. The individual had two copies of the APOE3 Christchurch (R136S) mutation, unusually high brain amyloid levels and limited tau and neurodegenerative measurements. Our findings have implications for the role of APOE in the pathogenesis, treatment and prevention of Alzheimer's disease.
Subramanian S, Maurer AC, Bator CM, Makhov AM, Conway JF, Turner KB, Marden JH, Vandenberghe LH, Hafenstein SL. Filling Adeno-Associated Virus Capsids: Estimating Success by Cryo-Electron Microscopy. Hum Gene Ther 2019;30(12):1449-1460.Abstract
Adeno-associated viruses (AAVs) have been employed successfully as gene therapy vectors in treating various genetic diseases for almost two decades. However, transgene packaging is usually imperfect, and developing a rapid and accurate method for measuring the proportion of DNA encapsidation is an important step for improving the downstream process of large scale vector production. In this study, we used two-dimensional class averages and three-dimensional classes, intermediate outputs in the single particle cryo-electron microscopy (cryo-EM) image reconstruction pipeline, to determine the proportion of DNA-packaged and empty capsid populations. Two different preparations of AAV3 were analyzed to estimate the minimum number of particles required to be sampled by cryo-EM in order for robust calculation of the proportion of the full versus empty capsids in any given sample. Cost analysis applied to the minimum amount of data required for a valid ratio suggests that cryo-EM is an effective approach to analyze vector preparations.
Yizhak K, Aguet F, Kim J, Hess JM, Kübler K, Grimsby J, Frazer R, Zhang H, Haradhvala NJ, Rosebrock D, Livitz D, Li X, Arich-Landkof E, Shoresh N, Stewart C, Segrè AV, Branton PA, Polak P, Ardlie KG, Getz G. RNA sequence analysis reveals macroscopic somatic clonal expansion across normal tissues. Science 2019;364(6444)Abstract
How somatic mutations accumulate in normal cells is poorly understood. A comprehensive analysis of RNA sequencing data from ~6700 samples across 29 normal tissues revealed multiple somatic variants, demonstrating that macroscopic clones can be found in many normal tissues. We found that sun-exposed skin, esophagus, and lung have a higher mutation burden than other tested tissues, which suggests that environmental factors can promote somatic mosaicism. Mutation burden was associated with both age and tissue-specific cell proliferation rate, highlighting that mutations accumulate over both time and number of cell divisions. Finally, normal tissues were found to harbor mutations in known cancer genes and hotspots. This study provides a broad view of macroscopic clonal expansion in human tissues, thus serving as a foundation for associating clonal expansion with environmental factors, aging, and risk of disease.
Wang SK, Xue Y, Rana P, Hong CM, Cepko CL. Soluble CX3CL1 gene therapy improves cone survival and function in mouse models of retinitis pigmentosa. Proc Natl Acad Sci U S A 2019;116(20):10140-10149.Abstract
Retinitis pigmentosa (RP) is a disease that initially presents as night blindness due to genetic deficits in the rod photoreceptors of the retina. Rods then die, causing dysfunction and death of cone photoreceptors, the cell type that mediates high acuity and color vision, ultimately leading to blindness. We investigated immune responses in mouse models of RP and found evidence of microglia activation throughout the period of cone degeneration. Using adeno-associated vectors (AAVs), delivery of genes encoding microglial regulatory signals led to the identification of AAV serotype 8 (AAV8) soluble CX3CL1 (sCX3CL1) as a promising therapy for degenerating cones. Subretinal injection of AAV8-sCX3CL1 significantly prolonged cone survival in three strains of RP mice. Rescue of cones was accompanied by improvements in visual function. AAV8-sCX3CL1 did not affect rod survival, microglia localization, or inflammatory cytokine levels in the retina. Furthermore, although RNA sequencing of microglia demonstrated marked transcriptional changes with AAV8-sCX3CL1, pharmacological depletion of up to ∼99% of microglia failed to abrogate the effect of AAV8-sCX3CL1 on cone survival. These findings indicate that AAV8-sCX3CL1 can rescue cones in multiple mouse models of RP via a pathway that does not require normal numbers of microglia. Gene therapy with sCX3CL1 is a promising mutation-independent approach to preserve vision in RP and potentially other forms of retinal degeneration.

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