Human Gene Therapy Trials Research Articles

Animal Models for the Treatment of Human Diseases – A Review

Abstract Currently, studies conducted on animal models provide new insight into the aetiology and course of various pathological conditions in humans. Due to the cause of disease, animal models can be divided into two categories: natural models, such as nude mice, and induced models, such as transgenic pigs. This article discusses several examples of animal models and their contributions to the development of effective therapies for human diseases such as muscular dystrophy, cystic fibrosis, and atherosclerosis. In addition, we presented the latest news on gene therapy, xenotransplantation, and drug trials in humans and animals.

High-Density Clonal Analysis Reveals Highly Active Contribution of Multipotent Hematopoietic Stem Cells during Early Phases of Hematopoietic Recovery after Transplantation

Recovery after conditioning and transplantation of hematopoietic stem and progenitor cells (HSPC) is thought to be biphasic, with short-term engrafting progenitors driving the recovery for 6-9 months and multipotent hematopoietic stem cells (HSCs) providing long-term repopulation. Recent clonal tracking data from autologous human gene therapy trials seems to support this model (Biasco et al. 2016, Cell Stem Cell; Six et al. 2020, Blood). These recent reports investigating the contribution of HSCs in patients are based on the longitudinal tracking of thousands of gene-marked cells using retroviral integration site analysis (ISA). While this technology is very reliable to follow gene therapy patients and monitor the potential outgrowth of dominant or malignant clones, low sensitivity and high error rates require significant data exclusion and sophisticated statistical tests to ensure data reliability (Adair et al. 2020, Molecular Therapy MCD). Lack of sensitivity can be overcome by increasing the frequency (high density) of sampling. However, limited material from patients remains a bottleneck for improved data quality and, consequently, correct interpretation of such complex datasets.To overcome the limitations of ISA and determine the onset of HSC contribution we performed high-density sampling for ISA in nonhuman primates (NHPs) transplanted with gene-modified HSCs. In the first month of hematopoietic recovery weekly blood samples were taken to enhance data density and increase the reliablity to detect clones with low abundance. Animals were followed up to 5 years to confirm that identified HSC clones persist long-term. Finally, clonal tracking data from the NHPs was used to inform a simulation of hematopoietic reconstitution, determine the temporal involvement of HSCs, and refine the phases of hematopoietic recovery after myeloablation and HSC transplantation.In contrast to the current biphasic model, contribution of multipotent HSCs clones was detected in the very first blood samples taken 2 to 3 weeks post-transplant during neutrophil recovery. HSC clones found in these early time points persisted long-term throughout the entire follow-up and were detected in bone marrow CD34 + cells 4 years later. Most surprisingly, multipotent HSCs became the dominant source for mature blood cells in the peripheral blood as early as 50 days post-transplant. To understand the observed kinetics of HSC contribution and change in clonal diversity in our dataset, we simulated the clonal outgrowth and differentiation of multipotent clones. Simulations predicted that hematopoietic recovery is primarily HSC driven and HSC contribution follows a stochastic pattern. Finally, to confirm that HSCs proliferation and differentiation is a stochastic process, in vitro experiments in colony-forming cell (CFC) assays were carried out. As predicted, the decision of individual HSCs to either grow into a larger pool or differentiate and get lost followed the same kinetics as observed in vivo.Here, we show evidence that long-term persisting multipotent HSCs actively contribute during early hematopoietic reconstitution after myeloablation and HSC transplantation. Enhanced sampling showed that multipotent HSCs produce neutrophils during recovery and become the predominant source of mature blood cells as early as 50 days post-transplant. Most importantly, observed changes in the clonal diversity during early recovery suggest a stochastic engraftment of HSCs rather than a bi-phasic reconstitution initially driven by short-term progenitors. These findings should have important implications for the design of ex vivo and in vivo HSC gene therapy and genome editing approaches. [Display omitted] DisclosuresRadtke: 47 Inc.: Consultancy; Ensoma Inc.: Consultancy. Kiem: Homology Medicines: Consultancy; VOR Biopharma: Consultancy; Ensoma Inc.: Consultancy, Current holder of individual stocks in a privately-held company.

An Inquiry into Life in the Universe (Vol. 25, No. 10, Full Issue)

For the month of October 2021, APBN explores astrobiology and the planetary sciences. In Features, we look into the age-old question: are we alone in the Universe? If not, how might life emerge elsewhere in the Cosmos and taking what we know now, where do we go from here? Then we have a special contribution by Professor Chandra Wickramasinghe, astronomer and pioneer of astrobiology, who discusses the Panspermia theory and the role of culture in delaying the acceptance of the theory. In Columns, we review the latest human gene therapy trials using adeno-associated viruses as vectors to treat neurodegenerative disorders. Finally, in Spotlights, we share highlights from a conference by SGInnovate and CATALYST potential of bioprinting in personalised medicine and an interview with Dr. Senthil Sockalingam, Head of IQVIA Biotech, JAPAC & Chief Medical Officer, APAC, on its expansion in the Asia-Pacific and how the company's new innovative approach will utilise data and analytics to accelerate clinical development.

Cell-Mediated Immunity to NAGLU Transgene Following Intracerebral Gene Therapy in Children With Mucopolysaccharidosis Type IIIB Syndrome.

Mucopolysaccharidosis type IIIB syndrome (Sanfilippo disease) is a rare autosomic recessif disorder caused by mutations in the α-N-acetylglucosaminidase (NAGLU) gene coding for a lysosomal enzyme, leading to neurodegeneration and progressive deterioration of cognitive abilities in affected children. To supply the missing enzyme, several recent human gene therapy trials relied on the deposit of adeno-associated virus (AAV) vectors directly into the brain. We reported safety and efficacy of an intracerebral therapy in a phase 1/2 clinical trial (https://clinicaltrials.gov/ct2/show/NCT03300453), with a recombinant AAV serotype 2/5 (rAAV2/5) coding human NAGLU in four children with MPS IIIB syndrome receiving immunosuppression. It was reported that AAV-mediated gene therapies might elicit a strong host immune response resulting in decreased transgene expression. To address this issue, we performed a comprehensive analysis of cellular immunity and cytokine patterns generated against the therapeutic enzyme in the four treated children over 5.5 years of follow-up. We report the emergence of memory and polyfunctional CD4+ and CD8+ T lymphocytes sensitized to the transgene soon after the start of therapy, and appearing in peripheral blood in waves throughout the follow-up. However, this response had no apparent impact on CNS transgene expression, which remained stable 66 months after surgery, possibly a consequence of the long-term immunosuppressive treatment. We also report that gene therapy did not trigger neuroinflammation, evaluated through the expression of cytokines and chemokines in patients’ CSF. Milder disease progression in the youngest patient was found associated with low level and less differentiated circulating NAGLU-specific T cells, together with the lack of proinflammatory cytokines in the CSF. Findings in this study support a systematic and comprehensive immunomonitoring approach for understanding the impact immune reactions might have on treatment safety and efficacy of gene therapies.

Gene therapy in color vision deficiency: a review.

Color vision deficiencies are a group of vision disorders, characterized by abnormal color discrimination. They include red-green color blindness, yellow-blue color blindness and achromatopsia, among others. The deficiencies are caused by mutations in the genes coding for various components of retinal cones. Gene therapy is rising as a promising therapeutic modality. The purpose of this review article is to explore the available literature on gene therapy in the different forms of color vision deficiencies. A thorough literature review was performed on PubMed using the keywords: color vision deficiencies, gene therapy, achromatopsia and the various genes responsible for this condition (OPN1LW, OPN1MW, ATF6, CNGA3, CNGB3, GNAT2, PDE6H, and PDE6C). Various adenovirus vectors have been deployed to test the efficacy of gene therapy for achromatopsia in animals and humans. Gene therapy trials in humans and animals targeting mutations in CNGA3 have been performed, demonstrating an improvement in electroretinogram (ERG)-investigated cone cell functionality. Similar outcomes have been reported for experimental studies on other genes (CNGB3, GNAT2, M- and L-opsin). It has also been reported that delivering the genes via intravitreal rather than subretinal injections could be safer. There are currently 3 ongoing human clinical trials for the treatment of achromatopsia due to mutations in CNGB3 and CNGA3. Experimental studies and clinical trials generally showed improvement in ERG-investigated cone cell functionality and visually elicited behavior. Gene therapy is a promising novel therapeutic modality in color vision deficiencies.

A Quantitative Chloride Channel Conductance Assay for Efficacy Testing of AAV.BEST1.

Mutations in the human BEST1 gene are responsible for a number of distinct retinal disorders known as bestrophinopathies, for which there are no current treatments. The protein product, bestrophin-1, is expressed in the retinal pigment epithelium (RPE) where it localizes to the basolateral membrane and acts as a Ca2+-activated chloride channel. Recent studies have shown successful BEST1-mediated gene transfer to the RPE, indicating human clinical trials of BEST1 gene therapy may be on the horizon. A critical aspect of such trials is the ability to assess the efficacy of vector prior to patient administration. Here, an assay is presented that enables the quantitative assessment of AAV-mediated BEST1 chloride conductance as a measure of vector efficacy. Expression of BEST1 following transduction of HEK293 cells with AAV.BEST1 vectors was confirmed by liquid chromatography, Western blot, and immunocytochemistry. Whole-cell patch-clamp showed increased chloride conductance in BEST1-transduced cells compared to sham-transduced and untransduced controls. Exogenous chloride current correlated to BEST1 expression level, with an enhanced AAV.BEST1.WPRE vector providing higher expression levels of BEST1 and increases in chloride conductance. This study presents in vitro electrophysical quantification of bestrophin-1 following AAV-mediated gene transfer, providing vital functional data on an AAV gene therapy product that will support a future application for regulatory approval.

Genome Editing Technologies: From Bench Side to Bedside

The development of genome editing technologies has given the chance to researchers to manipulate any genomic sequences precisely. This ability is very useful for creating animal models to study human diseases in vivo; for easy creation of isogenic cell lines to study in vitro and most importantly for overcoming many disadvantages that the researchers faced during the human gene therapy trials. Here we review the basic mechanisms of genome editing technology and the four genome-editing platforms. We also discuss the applications of these novel technologies in preclinical and clinical studies in four groups according to the mechanism used, and lastly, summarize the problems in these technologies.

A cure in the womb

Emboldened by recent success stories of human gene therapy trials in children and infants, researchers are now pushing to treat diseases before birth. Some scientists are excited by the idea of using fetal gene therapy to fix genetic conditions that can’t be treated after birth, but others raise questions about the technique’s feasibility and practicality in humans. A newly published study demonstrates that surgically injecting DNA-stuffed viruses into mice can treat genetic diseases in the womb, when the potential for improvement is the most dramatic. It shows that the surgery is possible in fetal monkeys as well. “For many genetic diseases, the damage starts early,” says Simon N. Waddington, a researcher at University College London who led the new study on an experimental gene therapy for Gaucher disease. The disease is the most common lysosomal storage disorder, a group of metabolic conditions in which an enzyme deficiency results in toxic

Genotoxic Lemons Become Epigenomic Lemonade.

Active regulatory elements in hematopoietic stem cells (HSCs) are incompletely characterized, since extant approaches immunophenotypically define and isolate rare HSCs. In the current issue of Cell Stem Cell, Wünsche etal. (2018) use γ-retroviral insertion sites from a human gene therapy trial to identify the active enhancer landscape of functionally characterized HSCs.

Gene therapies development: slow progress and promising prospect

ABSTRACTBackground: In 1989, the concept of human gene therapies has emerged with the first approved human gene therapy trial of Rosenberg et al. Gene therapies are considered as promising therapies applicable to a broad range of diseases.Objective: The objective of this study was to review the descriptive data on gene therapy clinical trials conducted worldwide between 1989 and 2015, and to discuss potential success rates of these trials over time and anticipated market launch in the upcoming years.Methods: A publicly available database, ‘Gene Therapy Clinical Trials Worldwide’, was used to extract descriptive data on gene therapy clinical trials: (1) number of trials per year between 1989 and 2015; (2) countries; (3) diseases targeted by gene therapies; (4) vectors used for gene delivery; (5) trials status; (6) phases of development.Results: Between 1989 and 2015, 2,335 gene therapy clinical trials have been completed, were ongoing or approved (but not started) worldwide. The number of clinical trials did not increase steadily over time; it reached its highest peak in 2015 (163 trials). Almost 95% of the trials were in early phases of development and 72% were ongoing. The United States undertook 67% of gene therapy clinical trials. The majority of gene therapies clinical trials identified targeted cancer diseases.Conclusion: The first gene therapy was approved in the European Union in 2012, after two decades of dashed expectations. This approval boosted the investment in developing gene therapies. Regulators are creating a specific path for rapid access of those new therapies, providing hope for manufacturers, healthcare professionals, and patients. However, payers are increasingly scrutinizing the additional benefits of the new therapies. Major steps forward are expected in the field of gene therapies in the future.

The long terminal repeat negative control region is a critical element for insertional oncogenesis after gene transfer into hematopoietic progenitors with Moloney murine leukemia viral vectors

Integrating vectors based on γ-retroviruses and containing full-length long terminal repeats (LTRs) have been associated with activation of oncogene expression and leukemogenesis in human gene therapy trials. Identification of the specific molecular elements of the LTRs that have a role in insertional oncogenesis events is important as it can lead to the development of safer gene transfer vectors. The negative control region (NCR) of the LTR is a particularly well-conserved sequence among mammalian γ-retroviruses with demonstrated regulatory activity of gene transcription in hematopoietic cells, which led us to hypothesize that this region may have a role in insertional oncogenesis after γ-retroviral vector (GV)-mediated gene transfer into hematopoietic progenitors. We used an in vitro assay of murine bone marrow cell immortalization to compare the immortalization capabilities of a series of GVs carrying murine leukemia virus (MLV) LTR deletion mutants. Compared with GV carrying the full-length MLV LTR, deletion of the complete LTR enhancer sequence showed significant reduction of immortalization rates. However, the use of a mutant LTR deleted of the enhancer sequence, with exception of the NCR, did not affect immortalization. Importantly, the inclusion of an LTR mutant devoid only of the NCR did show significant reduction of immortalization rates compared with the full LTR sequence. Therefore, our data point to the NCR as a key element for immortalization and justify additional studies to evaluate its specific role in MLV-mediated insertional oncogenesis.

Current status of ex vivo gene therapy for hematological disorders: a review of clinical trials in Japan around the world.

Gene therapies are classified into two major categories, namely, in vivo and ex vivo. Clinical trials of human gene therapy began with the ex vivo techniques. Based on the initial successes of gene-therapy clinical trials, these approaches have spread worldwide. The number of gene therapy trials approved worldwide increased gradually starting in 1989, reaching 116 protocols per year in 1999, and a total of 2210 protocols had been approved by 2015. Accumulating clinical evidence has demonstrated the safety and benefits of several types of gene therapy, with the exception of serious adverse events in several clinical trials. These painful experiences were translated backward to basic science, resulting in the development of several new technologies that have influenced the recent development of ex vivo gene therapy in this field. To date, six gene therapies have been approved in a limited number of countries worldwide. In Japan, clinical trials of gene therapy have developed under the strong influence of trials in the US and Europe. Since the initial stages, 50 clinical trials have been approved by the Japanese government. In this review, the history and current status of clinical trials of ex vivo gene therapy for hematological disorders are introduced and discussed.

281. Enforced Expression of a Mutant HMGA2 Gene Leads to Competitive Expansion and High Level Marking of Long-Term Hematopoietic Stem Cells in Transplanted Nemestrina Macaques

An important goal in gene therapy and stem cell transplant is to expand hematopoietic stem cells (HSCs) without causing hematopoietic abnormalities. This is particularly challenging in humans and other primates, where expansion protocols that work in mouse models are often less effective in primate HSCs. One approach is to test genes that have led to clonal expansions by vector insertion site effects in human gene therapy trials. In this study, we recreated a mutant HMGA2 cDNA that was associated with a benign clonal expansion in a patient treated on a β-thalassemia gene therapy trial. A lentiviral vector was generated that expressed a cDNA for human HMGA2 with a 3’ UTR deletion that eliminates all seven let-7 microRNA binding sites and thereby avoids the known repressive effects of let-7 miRNAs on HMGA2 expression. A gamma-retroviral MSCV promoter was used to express this mutant HMGA2 cDNA along with an Ires-GFP cassette. Bone marrow CD34+ cells were collected for Nemestrina Macaques and half the cells were transduced with the lentiviral vector containing the HMGA2-GFP construct while the other half was transduced with a mCherry control vector. The transduction efficiencies were approximately the same prior to transplant. At three months post transplantation, the marking in the peripheral blood mononuclear cells was 2.9% GFP+ and 1.1% mCherry+ for animal #16, and was 2.7% GFP+ and 3.2% mCherry+ for animal #27, suggesting relatively equivalent and low levels of transduction of repopulating cells. The HMGA2-GFP marking progressively increased over 21 and 26 months in the peripheral blood leukocytes to 39% for #16 and 41% for #27 while the mCherry marked cells have decreased (Fig 1AFig 1A). Equivalent levels of marking were seen in various mature peripheral blood lineages, including circulating erythyrocytes, suggesting that expansion had occurred in pluripotent HSCs. This HSC expansion was further demonstrated by a marking analysis in bone marrow cells that showed 44.6% and 75.2% GFP marking in the CD34+CD45RA- HSC compartment for animal #16 and #27 respectively, at the latest time point. Clonality analyses using vector integration sites (VIS) showed overall oligoclonal marking in both the GFP+ and mCherry+ cells in both animals (Fig 1BFig 1B). The top most frequent VISs were present in all PB lineages indicating that expansion had occurred in multiple HSC clones. The WBC counts, lineage distribution in the peripheral blood, the percentage of CD34+ cells in the bone marrow and all the mature lineages in the peripheral blood are all within the normal range, demonstrating lack of any detectable hematopoietic abnormality. Gene expression microarray analysis of RNA from sorted BM CD34+GFP+ and CD34+mCherry+ cells showed sharp upregulation of several genes in the HMGA2-expressing cells, particularly the IGF2BP gene, a known downstream target of HMGA2. In summary, our data show that long-term HSCs from non-human primates can be progressively expanded in vivo over several years by overexpressing HMGA2. This could be useful for gene therapy, particularly for expanding and obtaining high numbers of transduced erythrocytes and granulocytes for diseases in which a naturally occurring selection advantage is not present.View Large Image | Download PowerPoint Slide

282. Circumventing Hematopoietic Toxicities and Reversing Sickle Phenotype by Lineage-Specific BCL11A Knock Down and γ-Globin Induction

The fetal hemoglobin repressor BCL11A represents a therapeutic target for β-hemoglobinopathies as reduced expression of BCL11A leads to simultaneous increased γ-globin and reduced β-globin expression. Reversing this hemoglobin switch is particularly relevant in sickle cell disease to reduce the βS concentration and increase expression of the protective fetal hemoglobin (HbF, α2γ2). Here we show that despite use of optimized shRNAs embedded into a miRNA (shRNAmiR) architecture to reduce non-specific cellular toxicities (Guda et al. MT, 2015), the knockdown of BCL11A profoundly and specifically impaired long-term engraftment of both human and mouse hematopoietic stem cells (HSCs). In competitive transplantation assays cells transduced with shRNAmiRs targeting the BCL11A mRNA were consistently underrepresented after hematopoietic reconstitution with gene modified HSCs. Although this effect was particularly pronounced in the B-cell compartment, it was also the case for all other assessed hematopoietic lineages. Mechanistically, while knock-down of BCL11A did not lead to a detectable phenotype in terms of apoptosis, growth or differentiation in human or mouse HSCs in vitro, a significant increase in S/G2-phase human HSCs after engraftment into NSG mice was found, a phenotype associated with stem cell exhaustion. To avoid this BCL11A-specific HSC toxicity, we suppressed BCL11A in erythroid cells in a lineage-specific fashion by using transcriptional regulatory elements derived from the β-globin locus. Utilizing this approach for the expression of the optimized shRNAmiRs led to stable long-term engraftment of mouse and human gene modified cells in congenic or NSG-mice, respectively. Transduced primary normal or sickle cell disease (SCD) human HSCs gave rise to erythroid cells with up to 90% reduction of BCL11A protein. These erythrocytes demonstrated 60-70% γ-chain expression and a corresponding increase in HbF at low vector copy numbers per cell (VCN<1.5). Similar results were obtained after in vitro differentiation of CD34+ cells harvested 16 weeks following engraftment in NSG mice. Transplantation of gene modified murine HSCs from BERK sickle cell mice led to a substantial improvement of sickle-associated hemolytic anemia and reticulocytosis, key phenotypes of SCD. In summary, we have shown an unexpected and severe toxicity of BCL11A knockdown in repopulating HSCs that has direct and important consequences for translation into human gene therapy trials. By utilizing erythroid lineage-specific and microRNA embedded expression of targeting shRNAs we demonstrate the capacity of lentivirus vectors to effect significant γ-globin induction leading to clinically relevant increases in HbF while obviating HSC toxicity.

363. Improvement of Sandhoff Phenotype Following Intravenous Injection of Adeno-Associated Viral Vector Expressing a Hexosaminidase Isoenzyme in Adult Sandhoff Mice: Preclinical Safety and Efficacy Study

GM2 gangliosidosis disorders stem from a Hexosaminidase A (HexA) isoenzyme deficiency. In humans, HexA is the sole enzyme able to catabolize GM2 ganglioside (GM2). The inability to effectively catabolize GM2 leads to neurodegeneration of the central nervous system. HexA is comprised of 2 subunits (α, β) and works with the GM2 activator protein (GM2AP). In the recent work by Tropak et al. (Mol Ther Met Clin Dev, in press), a hybrid subunit, named µ-subunit, was created (patent pending) by combining the stabilization and GM2AP binding sites of the β-subunit while conserving the catalytic properties of the α-subunit. The ‘µ’-subunit, coded by HEXM, can homodimerize and form a stable, functional enzyme, named HexM, which can interact with GM2AP to hydrolyse GM2. Previous work for successful correction of Sandhoff mice using AAV was only shown in neonatal mice (with immature blood-brain barrier (BBB)) and may not directly help in designing a human clinical trial. In the current study, we examined the efficacy/safety of IV injections of the scAAV9/HEXM vector at two doses in adult SD mice (with mature BBB). In addition, we also tested if an adjunct IV injection of mannitol provides any enhancement in efficacy. At 6 weeks old, the vector was injected via tail vein in cohorts of n=17 and n=15 SD mice at 2.5E+12 or 1.0E+13 vg/mouse, respectively. Another cohort of 16 mice received IV mannitol (3g/kg) prior to an IV injection of 2.5E+12 vg scAAV9/HEXM. Some mice from low dose group were euthanized at 16 weeks for direct analysis with untreated SD control mice, while the remainder were left until for terminal survival. Analysis of survival benefit, locomotor behaviour, biochemical and molecular parameters were performed. While untreated SD mice had a 16 week humane endpoint, 4 of 7 mice in higher dose group are now surviving past 56 weeks, 1 of 12 mice in the low dose cohort, and 4 of 9 mice in the mannitol cohort are surviving past 52 weeks. These increases in survival are all highly significant compared to the ~16 week humane endpoint of untreated SD mice. Behaviourally, there are no major significant differences in locomotion between the groups until after 15 weeks, when the adjunct mannitol group significantly outperforms the PBS group. Survival and behaviour monitoring, and the biochemical analyses for this study are ongoing. The preliminary results from this study show delayed onset of the SD phenotype with a single AAV9/HEXM injection and a significant benefit of a pre-injection of IV mannitol. This study is the first to show that an IV gene transfer using a scAAV/HEXM vector can provide survival and behavioural benefit in adult SD mice especially with adjunct use of mannitol. We propose that these results can advise the design of a human gene therapy trial for SD and the related Tay-Sachs disease.

Preclinical Dose-Escalation Study of Intravitreal AAV-RS1 Gene Therapy in a Mouse Model of X-linked Retinoschisis: Dose-Dependent Expression and Improved Retinal Structure and Function.

Gene therapy for inherited retinal diseases has been shown to ameliorate functional and structural defects in both animal models and in human clinical trials. X-linked retinoschisis (XLRS) is an early-age onset macular dystrophy resulting from loss of an extracellular matrix protein (RS1). In preparation for a human clinical gene therapy trial, we conducted a dose-range efficacy study of the clinical vector, a self-complementary AAV delivering a human retinoschisin (RS1) gene under control of the RS1 promoter and an interphotoreceptor binding protein enhancer (AAV8-scRS/IRBPhRS), in the retinoschisin knockout (Rs1-KO) mouse. The therapeutic vector at 1 × 10(6) to 2.5 × 10(9) (1E6-2.5E9) vector genomes (vg)/eye or vehicle was administered to one eye of 229 male Rs1-KO mice by intravitreal injection at 22 ± 3 days postnatal age (PN). Analysis of retinal function (dark-adapted electroretinogram, ERG), structure (cavities and outer nuclear layer thickness) by in vivo retinal imaging using optical coherence tomography, and retinal immunohistochemistry (IHC) for RS1 was done 3-4 months and/or 6-9 months postinjection (PI). RS1 IHC staining was dose dependent across doses ≥1E7 vg/eye, and the threshold for significant improvement in all measures of retinal structure and function was 1E8 vg/eye. Higher doses, however, did not produce additional improvement. At all doses showing efficacy, RS1 staining in Rs1-KO mouse was less than that in wild-type mice. Improvement in the ERG and RS1 staining was unchanged or greater at 6-9 months than at 3-4 months PI. This study demonstrates that vitreal administration of AAV8 scRS/IRBPhRS produces significant improvement in retinal structure and function in the mouse model of XLRS over a vector dose range that can be extended to a human trial. It indicates that a fully normal level of RS1 expression is not necessary for a therapeutic effect.

Development of rAAV2-CFTR: History of the First rAAV Vector Product to be Used in Humans.

The first human gene therapy trials using recombinant adeno-associated virus (rAAV) vectors were performed in cystic fibrosis (CF) patients. Over 100 CF patients were enrolled in 5 separate trials of rAAV2-CFTR administration via nasal, endobronchial, maxillary sinus, and aerosol delivery. Recombinant AAV vectors were designed to deliver the CF transmembrane regulator (CFTR) gene and correct the basic CFTR defect by restoring chloride transport and reverting the upregulation of proinflammatory cytokines. However, vector DNA expression was limited in duration because of the low incidence of integration and natural airway epithelium turnover. In addition, repeated administration of AAV-CFTR vector resulted in a humoral immune response that prevented effective gene transfer from subsequent doses of vector. AAV serotype 2 was used in human trials before the comparison with other serotypes and determination that serotypes 1 and 5 not only possess higher tropism for the airway epithelium, but also are capable of bypassing the binding and trafficking processes-both were important hindrances to the effectiveness of rAAV2. Although rAAV-CFTR gene therapy does not appear likely to supplant newer small-molecule CFTR modulators in the near future, early work with rAAV-CFTR provided an important foundation for later use of rAAV in humans.

Using a Simulation to Illustrate Crosscutting Concepts through a Disease Model

Using Pompe disease as a context affords the opportunity for students to consider multiple biological concepts and embraces the Next Generation Science Standards Disciplinary Core Ideas Structure and Function (LS1.A) and Inheritance of Traits (LS3.A) as well as Crosscutting Concepts Structure and Function and Cause and Effect. These crosscutting concepts are very much interrelated as we consider progression of disease from the molecular to the organismal level. The concepts are repeatedly emphasized, providing “explicit instructional support” for students to “develop a cumulative, coherent, and usable understanding of science and engineering.” DNA, proteins, enzymes, genetics, and human disease are taught together through the story of patients with Pompe disease as students engage in a simulated clinical assay and genetic analysis and present their findings in grand rounds. The activity is one of multiple lessons sequenced to scaffold student understanding of clinical and translational science, starting with a first-person perspective of a father who loses his infant son to Pompe and concluding with a role play based on actual events surrounding approval of human clinical trials of gene therapy for Pompe disease.

Retinal Development in Infants and Young Children with Achromatopsia

Retinal Development in Infants and Young Children with Achromatopsia

608. Evaluation of Pre-Existing Neutralizing Antibodies Against Tyrosine-Mutant AAV2 in Leber Hereditary Optic Neuropathy (LHON) Patients

PURPOSE: Pre-existing neutralizing antibodies against adeno-associated virus (AAV), due to natural AAV infections could interfere with the recombinant AAV vector-mediated gene transfer, thus decreasing the efficiency and preventing effective vector readministration. Here, we measured neutralizing antibody (NAb) titers against tyrosine-mutant AAV serotype 2 in Leber Hereditary Optic Neuropathy (LHON) patients.METHODS: Serum samples from LHON patients (n=53, age =33 + 1.9 yrs, average + se) were screened for the existence of NAbs to AAV2 using an invitro assay. For this, a self-complimentary AAV2(Y444,500,703F)-smCBA-mCherry vector was pre-incubated with heat inactivated LHON patient serum samples at various dilutions, beginning with a 1:5 dilution and ending at 1:20,480. The serum-vector mixtures were used to infect RGC5 cells at multiplicity of infection of 5000 vector genomes/per cell. Two days post-infection, cell viability was confirmed by microscopy and transduction scored by flow cytometry. Expression of mCherry was quantified by multiplying the percentage of cells positive for mCherry by the mean fluorescence intensity. The neutralizing antibody (NAb) titer was reported as the highest serum dilution that inhibited self-complementary AAV2 (Y444,500,703F)-smCBA-mCherry transduction >50% compared to no serum control.RESULTS: We found that the prevalence of NAbs to AAV2 (Y444,500,703F) among the LHON subjects was variable with majority, 49% (26/53) showing low NAb titers ( 0.05).CONCLUSIONS: Previous AAV2 exposure among LHON patients is low to high and these findings as they relate to the ongoing clinical human gene therapy trial (NCT02161380) will be discussed.