IU Indianapolis ScholarWorks :: Browsing by Subject "AAV"

Browsing by Subject "AAV"

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B Cell Depletion Eliminates FVIII Memory B Cells and Enhances AAV8-coF8 Immune Tolerance Induction When Combined With Rapamycin
(Frontiers, 2020-06-24) Biswas, Moanaro; Palaschak, Brett; Kumar, Sandeep R. P.; Rana, Jyoti; Markusic, David M.; Pediatrics, School of Medicine
Hemophilia A is an inherited coagulation disorder resulting in the loss of functional clotting factor VIII (FVIII). Presently, the most effective treatment is prophylactic protein replacement therapy. However, this requires frequent life-long intravenous infusions of plasma derived or recombinant clotting factors and is not a cure. A major complication is the development of inhibitory antibodies that nullify the replacement factor. Immune tolerance induction (ITI) therapy to reverse inhibitors can last from months to years, requires daily or every other day infusions of supraphysiological levels of FVIII and is effective in only up to 70% of hemophilia A patients. Preclinical and recent clinical studies have shown that gene replacement therapy with AAV vectors can effectively cure hemophilia A patients. However, it is unclear how hemophilia patients with high risk inhibitor F8 mutations or with established inhibitors will respond to gene therapy, as these patients have been excluded from ongoing clinical trials. AAV8-coF8 gene transfer in naïve BALB/c-F8e16−/Y mice (BALB/c-HA) results in anti-FVIII IgG1 inhibitors following gene transfer, which can be prevented by transient immune modulation with anti-mCD20 (18B12) and oral rapamycin. We investigated if we could improve ITI in inhibitor positive mice by combining anti-mCD20 and rapamycin with AAV8-coF8 gene therapy. Our hypothesis was that continuous expression of FVIII protein from gene transfer compared to transient FVIII from weekly protein therapy, would enhance regulatory T cell induction and promote deletion of FVIII reactive B cells, following reconstitution. Mice that received anti-CD20 had a sharp decline in inhibitors, which corresponded to FVIII memory B (Bmem) cell deletion. Importantly, only mice receiving both anti-mCD20 and rapamycin failed to increase inhibitors following rechallenge with intravenous FVIII protein therapy. Our data show that B and T cell immune modulation complements AAV8-coF8 gene therapy in naïve and inhibitor positive hemophilia A mice and suggest that such protocols should be considered for AAV gene therapy in high risk or inhibitor positive hemophilia patients.
Chemical Modifications of the Capsid for Redirecting and Improving the Efficacy of Adeno-Associated Virus Vectors
(Mary Ann Liebert, 2021-12) Lam, Anh Kim; Frabutt, Dylan A.; Li, Lei; Xiao, Weidong; Pediatrics, School of Medicine
Adeno-associated virus (AAV) vector-directed gene therapy is one of the most exciting modalities of biotechnology as more applications enter clinical stage. Although AAV vectors generally feature low toxicity, high stability, and long-lasting transgene expression, potential challenging issues of AAV include high vector dose, limited tissue tropism, and the host immune response and inflammation, which are all related to the capsid protein. To overcome these challenges, various strategies have been developed to engineer AAV capsids. Apart from widely employed genetic engineering of capsid protein, powerful and versatile chemical modification strategies are underexploited. This minireview summarizes recent advances and our perspectives for future direction in AAV capsid chemical modification to enhance its therapeutic use for gene therapy.
Exogenous Oncostatin M Induces Cardiac Dysfunction, Musculoskeletal Atrophy, and Fibrosis
(Elsevier, 2022) Jengelley, Daenique H. A.; Wang, Meijing; Narasimhan, Ashok; Rupert, Joseph E.; Young, Andrew R.; Zhong, Xiaoling; Horan, Daniel J.; Robling, Alexander G.; Koniaris, Leonidas G.; Zimmers, Teresa A.; Biochemistry and Molecular Biology, School of Medicine
Musculoskeletal diseases such as muscular dystrophy, cachexia, osteoarthritis, and rheumatoid arthritis impair overall physical health and reduce survival. Patients suffer from pain, dysfunction, and dysmobility due to inflammation and fibrosis in bones, muscles, and joints, both locally and systemically. The Interleukin-6 (IL-6) family of cytokines, most notably IL-6, is implicated in musculoskeletal disorders and cachexia. Here we show elevated circulating levels of OSM in murine pancreatic cancer cachexia and evaluate the effects of the IL-6 family member, Oncostatin M (OSM), on muscle and bone using adeno-associated virus (AAV) mediated over-expression of murine OSM in wildtype and IL-6 deficient mice. Initial studies with high titer AAV-OSM injection yielded high circulating OSM and IL-6, thrombocytosis, inflammation, and 60% mortality without muscle loss within 4 days. Subsequently, to mimic OSM levels in cachexia, a lower titer of AAV-OSM was used in wildtype and Il6 null mice, observing effects out to 4 weeks and 12 weeks. AAV-OSM caused muscle atrophy and fibrosis in the gastrocnemius, tibialis anterior, and quadriceps of the injected limb, but these effects were not observed on the non-injected side. In contrast, OSM induced both local and distant trabecular bone loss as shown by reduced bone volume, trabecular number, and thickness, and increased trabecular separation. OSM caused cardiac dysfunction including reduced ejection fraction and reduced fractional shortening. RNA-sequencing of cardiac muscle revealed upregulation of genes related to inflammation and fibrosis. None of these effects were different in IL-6 knockout mice. Thus, OSM induces local muscle atrophy, systemic bone loss, tissue fibrosis, and cardiac dysfunction independently of IL-6, suggesting a role for OSM in musculoskeletal conditions with these characteristics, including cancer cachexia.
Experimental Variables that Affect Human Hepatocyte AAV Transduction in Liver Chimeric Mice
(Elsevier, 2020-09-11) Zou, Chenhui; Vercauteren, Koen O. A.; Michailidis, Eleftherios; Kabbani, Mohammad; Zoluthkin, Irene; Quirk, Corrine; Chiriboga, Luis; Yazicioglu, Mustafa; Anguela, Xavier M.; Meuleman, Philip; High, Katherine A.; Herzog, Roland W.; Jong, Ype P. de; Pediatrics, School of Medicine
Adeno-associated virus (AAV) vector serotypes vary in their ability to transduce hepatocytes from different species. Chimeric mouse models harboring human hepatocytes have shown translational promise for liver-directed gene therapies. However, many variables that influence human hepatocyte transduction and transgene expression in such models remain poorly defined. Here, we aimed to test whether three experimental conditions influence AAV transgene expression in immunodeficient, fumaryl-acetoactetate-hydrolase-deficient (Fah−/−) chimeric mice repopulated with primary human hepatocytes. We examined the effects of the murine liver injury cycle, human donor variability, and vector doses on hepatocyte transduction with various AAV serotypes expressing a green fluorescent protein (GFP). We determined that the timing of AAV vector challenge in the liver injury cycle resulted in up to 7-fold differences in the percentage of GFP expressing human hepatocytes. The GFP+ hepatocyte frequency varied 7-fold between human donors without, however, changing the relative transduction efficiency between serotypes for an individual donor. There was also a clear relationship between AAV vector doses and human hepatocyte transduction and transgene expression. We conclude that several experimental variables substantially affect human hepatocyte transduction in the Fah−/− chimera model, attention to which may improve reproducibility between findings from different laboratories.
Feasibility of using NF1-GRD and AAV for gene replacement therapy in NF1-associated tumors
(Springer Nature, 2019-06) Bai, Ren-Yuan; Esposito, Dominic; Tam, Ada J.; McCormick, Frank; Riggins, Gregory J.; Clapp, D. Wade; Staedtke, Verena; Pediatrics, School of Medicine
Neurofibromatosis type 1, including the highly aggressive malignant peripheral nerve sheath tumors (MPNSTs), is featured by the loss of functional neurofibromin 1 (NF1) protein resulting from genetic alterations. A major function of NF1 is suppressing Ras activities, which is conveyed by an intrinsic GTPase-activating protein-related domain (GRD). In this study, we explored the feasibility of restoring Ras GTPase via exogenous expression of various GRD constructs, via gene delivery using a panel of adeno-associated virus (AAV) vectors in MPNST and human Schwann cells (HSCs). We demonstrated that several AAV serotypes achieved favorable transduction efficacies in those cells and a membrane-targeting GRD fused with an H-Ras C-terminal motif (C10) dramatically inhibited the Ras pathway and MPNST cells in a NF1-specific manner. Our results opened up a venue of gene replacement therapy in NF1-related tumors.
Gene therapy – are we ready now?
(Wiley, 2022) Kaczmarek, Radoslaw; Pediatrics, School of Medicine
Introduction: Haemophilia therapy has evolved from rudimentary transfusion-based approaches to an unprecedented level of innovation with glimmers of functional cure brought by gene therapy. After decades of misfires, gene therapy has normalized factor (F)VIII and factor (F)IX levels in some individuals in the long term. Several clinical programmes testing adeno-associated viral (AAV) vector gene therapy are approaching completion with imminent regulatory approvals. Discussion: Phase 3 studies along with multiyear follow-up in earlier phase investigations raised questions about efficacy as well as short- and long-term safety, prompting a reappraisal of AAV vector gene therapy. Liver toxicities, albeit mostly low-grade, occur in the first year in at least some individuals in all haemophilia A and B trials and are poorly understood. Extreme variability and unpredictability of outcome, as well as a slow decline in factor expression (seemingly unique to FVIII gene therapy), are vexing because immune responses to AAV vectors preclude repeat dosing, which could increase suboptimal or restore declining expression, while overexpression may result in phenotoxicity. The long-term safety will need lifelong monitoring because AAV vectors, contrary to conventional wisdom, integrate into chromosomes at the rate that calls for vigilance. Conclusions: AAV transduction and transgene expression engage the host immune system, cellular DNA processing, transcription and translation machineries in ways that have been only cursorily studied in the clinic. Delineating those mechanisms will be key to finding mitigants and solutions to the remaining problems, and including individuals who cannot avail of gene therapy at this time.
Homer2 within the nucleus accumbens core bidirectionally regulates alcohol intake by both P and Wistar rats
(Elsevier, 2015-09) Haider, Arshad; Woodward, Nicholas C.; Lominac, Kevin D.; Sacramento, Arianne D.; Klugmann, Matthias; Bell, Richard L.; Szumlinski, Karen K.; Department of Psychiatry, IU School of Medicine
In murine models of alcoholism, the glutamate receptor scaffolding protein Homer2 bidirectionally regulates alcohol intake. Although chronic alcohol drinking increases Homer2 expression within the core subregion of the nucleus accumbens (NAc) of alcohol-preferring P rats, the relevance of this neuroadaptation for alcohol intake has yet to be determined in rats. Thus, the present study employed an adeno-associated viral vector (AAV) strategy to over-express and knock down the major rodent isoform Homer2b within the NAc of both P and outbred Wistar rats to examine for changes in alcohol preference and intake (0-30% v/v) under continuous-access procedures. The generalization of AAV effects to non-drug, palatable, sweet solutions was also determined in tests of sucrose (0-5% w/v) and saccharin (0-0.125% w/v) intake/preference. No net-flux in vivo microdialysis was conducted for glutamate in the NAc to relate Homer2-dependent changes in alcohol intake to extracellular levels of glutamate. Line differences were noted for sweet solution preference and intake, but these variables were not affected by intra-NAc AAV infusion in either line. In contrast, Homer2b over-expression elevated, while Homer2b knock-down reduced, alcohol intake in both lines, and this effect was greatest at the highest concentration. Strikingly, in P rats there was a direct association between changes in Homer2b expression and NAc extracellular glutamate levels, but this effect was not seen in Wistar rats. These data indicate that NAc Homer2b expression actively regulates alcohol consumption by rats, paralleling this previous observation in mice. Overall, these findings underscore the importance of mesocorticolimbic glutamate activity in alcohol abuse/dependence and suggest that Homer2b and/or its constituents may serve as molecular targets for the treatment of these disorders.
Immune Response Mechanisms against AAV Vectors in Animal Models
(Elsevier, 2020-06) Martino, Ashley T.; Markusic, David M.; Pediatrics, School of Medicine
Early preclinical studies in rodents and other species did not reveal that vector or transgene immunity would present a significant hurdle for sustained gene expression. While there was early evidence of mild immune responses to adeno-associated virus (AAV) in preclinical studies, it was generally believed that these responses were too weak and transient to negatively impact sustained transduction. However, translation of the cumulative success in treating hemophilia B in rodents and dogs with an AAV2-F9 vector to human studies was not as successful. Despite significant progress in recent clinical trials for hemophilia, new immunotoxicities to AAV and transgene are emerging in humans that require better animal models to assess and overcome these responses. The animal models designed to address these immune complications have provided critical information to assess how vector dose, vector capsid processing, vector genome, difference in serotypes, and variations in vector delivery route can impact immunity and to develop approaches for overcoming pre-existing immunity. Additionally, a comprehensive dissection of innate, adaptive, and regulatory responses to AAV vectors in preclinical studies has provided a framework that can be utilized for development of immunomodulatory therapies to overcome or bypass immune responses and for developing strategic approaches toward engineering stealth AAV vectors that can circumvent immunity.
Improved Tissue Repair in Articular Cartilage Defects in Vivo by rAAV-Mediated Overexpression of Human Fibroblast Growth Factor 2
(Elsevier, 2005-08-01) Cucchiarini, Magali; Madry, Henning; Ma, Chunyan; Thurn, Tanja; Zurakowski, David; Menger, Michael D.; Kohn, Dieter; Trippel, Stephen B.; Terwilliger, Ernest F.; Orthopaedic Surgery, School of Medicine
Therapeutic gene transfer into articular cartilage is a potential means to stimulate reparative activities in tissue lesions. We previously demonstrated that direct application of recombinant adeno-associated virus (rAAV) vectors to articular chondrocytes in their native matrix in situ as well as sites of tissue damage allowed for efficient and sustained reporter gene expression. Here we test the hypothesis that rAAV-mediated overexpression of fibroblast growth factor 2 (FGF-2), one candidate for enhancing the repair of cartilage lesions, would lead to the production of a biologically active factor that would facilitate the healing of articular cartilage defects. In vitro, FGF-2 production from an rAAV-delivered transgene was sufficient to stimulate chondrocyte proliferation over a prolonged period of time. In vivo, application of the therapeutic vector significantly improved the overall repair, filling, architecture, and cell morphology of osteochondral defects in rabbit knee joints. Differences in matrix synthesis were also observed, although not to the point of statistical significance. This process may further benefit from cosupplementation with other factors. These results provide a basis for rAAV application to sites of articular cartilage damage to deliver agents that promote tissue repair.
Minimal Essential Human Factor VIII Alterations Enhance Secretion and Gene Therapy Efficiency
(Elsevier, 2020-10-22) Cao, Wenjing; Dong, Biao; Horling, Franziska; Firrman, Jenni A.; Lengler, Johannes; Klugmann, Matthias; de la Rosa, Maurus; Wu, Wenman; Wang, Qizhao; Wei, Hongying; Moore, Andrea R.; Roberts, Sean A.; Booth, Carmen J.; Hoellriegl, Werner; Li, Dong; Konkle, Barbara; Miao, Carol; Reipert, Birgit M.; Scheiflinger, Friedrich; Rottensteiner, Hanspeter; Xiao, Weidong; Pediatrics, School of Medicine
One important limitation for achieving therapeutic expression of human factor VIII (FVIII) in hemophilia A gene therapy is inefficient secretion of the FVIII protein. Substitution of five amino acids in the A1 domain of human FVIII with the corresponding porcine FVIII residues generated a secretion-enhanced human FVIII variant termed B-domain-deleted (BDD)-FVIII-X5 that resulted in 8-fold higher FVIII activity levels in the supernatant of an in vitro cell-based assay system than seen with unmodified human BDD-FVIII. Analysis of purified recombinant BDD-FVIII-X5 and BDD-FVIII revealed similar specific activities for both proteins, indicating that the effect of the X5 alteration is confined to increased FVIII secretion. Intravenous delivery in FVIII-deficient mice of liver-targeted adeno-associated virus (AAV) vectors designed to express BDD-FVIII-X5 or BDD-FVIII achieved substantially higher plasma FVIII activity levels for BDD-FVIII-X5, even when highly efficient codon-optimized F8 nucleotide sequences were employed. A comprehensive immunogenicity assessment using in vitro stimulation assays and various in vivo preclinical models of hemophilia A demonstrated that the BDD-FVIII-X5 variant does not exhibit an increased immunogenicity risk compared to BDD-FVIII. In conclusion, BDD-FVIII-X5 is an effective FVIII variant molecule that can be further developed for use in gene- and protein-based therapeutics for patients with hemophilia A.

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