Browsing by Author "Duan, Dongsheng"

Now showing 1 - 5 of 5
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    B cell focused transient immune suppression protocol for efficient AAV readministration to the liver
    (Elsevier, 2024-02-20) Rana, Jyoti; Herzog, Roland W.; Muñoz-Melero, Maite; Yamada, Kentaro; Kumar, Sandeep R. P.; Lam, Anh K.; Markusic, David M.; Duan, Dongsheng; Terhorst, Cox; Byrne, Barry J.; Corti, Manuela; Biswas, Moanaro; Pediatrics, School of Medicine
    Adeno-associated virus (AAV) vectors are used for correcting multiple genetic disorders. Although the goal is to achieve lifelong correction with a single vector administration, the ability to redose would enable the extension of therapy in cases in which initial gene transfer is insufficient to achieve a lasting cure, episomal vector forms are lost in growing organs of pediatric patients, or transgene expression is diminished over time. However, AAV typically induces potent and long-lasting neutralizing antibodies (NAbs) against capsid that prevents re-administration. To prevent NAb formation in hepatic AAV8 gene transfer, we developed a transient B cell-targeting protocol using a combination of monoclonal Ab therapy against CD20 (for B cell depletion) and BAFF (to slow B cell repopulation). Initiation of immunosuppression before (rather than at the time of) vector administration and prolonged anti-BAFF treatment prevented immune responses against the transgene product and abrogated prolonged IgM formation. As a result, vector re-administration after immune reconstitution was highly effective. Interestingly, re-administration before the immune system had fully recovered achieved further elevated levels of transgene expression. Finally, this immunosuppression protocol reduced Ig-mediated AAV uptake by immune cell types with implications to reduce the risk of immunotoxicities in human gene therapy with AAV.
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    Cas9-specific immune responses compromise local and systemic AAV CRISPR therapy in multiple dystrophic canine models
    (Springer Nature, 2021-11-24) Hakim, Chady H.; Kumar, Sandeep R. P.; Pérez-López, Dennis O.; Wasala, Nalinda B.; Zhang, Dong; Yue, Yongping; Teixeira, James; Zhang, Keqing; Million, Emily D.; Nelson, Christopher E.; Metzger, Samantha; Han, Jin; Louderman, Jacqueline A.; Schmidt, Florian; Feng, Feng; Grimm, Dirk; Smith, Bruce F.; Yao, Gang; Yang, N. Nora; Gersbach, Charles A.; Chen, Shi-jie; Herzog, Roland W.; Duan, Dongsheng; Pediatrics, School of Medicine
    Adeno-associated virus (AAV)-mediated CRISPR-Cas9 editing holds promise to treat many diseases. The immune response to bacterial-derived Cas9 has been speculated as a hurdle for AAV-CRISPR therapy. However, immunological consequences of AAV-mediated Cas9 expression have thus far not been thoroughly investigated in large mammals. We evaluate Cas9-specific immune responses in canine models of Duchenne muscular dystrophy (DMD) following intramuscular and intravenous AAV-CRISPR therapy. Treatment results initially in robust dystrophin restoration in affected dogs but also induces muscle inflammation, and Cas9-specific humoral and cytotoxic T-lymphocyte (CTL) responses that are not prevented by the muscle-specific promoter and transient prednisolone immune suppression. In normal dogs, AAV-mediated Cas9 expression induces similar, though milder, immune responses. In contrast, other therapeutic (micro-dystrophin and SERCA2a) and reporter (alkaline phosphatase, AP) vectors result in persistent expression without inducing muscle inflammation. Our results suggest Cas9 immunity may represent a critical barrier for AAV-CRISPR therapy in large mammals.
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    Immune Responses to Muscle-Directed Adeno-Associated Viral Gene Transfer in Clinical Studies
    (Mary Ann Liebert, 2023) Kumar, Sandeep R. P.; Duan, Dongsheng; Herzog, Roland W.; Pediatrics, School of Medicine
    Muscle-directed gene therapy with adeno-associated viral (AAV) vectors is undergoing clinical development for treating neuromuscular disorders and for systemic delivery of therapeutic proteins. Although these approaches show considerable therapeutic benefits, they are also prone to induce potent immune responses against vector or transgene products owing to the immunogenic nature of the intramuscular delivery route, or the high doses required for systemic delivery to muscle. Major immunological concerns include antibody formation against viral capsid, complement activation, and cytotoxic T cell responses against capsid or transgene products. They can negate therapy and even lead to life-threatening immunotoxicities. Herein we review clinical observations and provide an outlook for how the field addresses these problems through a combination of vector engineering and immune modulation.
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    Redundancy in Innate Immune Pathways That Promote CD8+ T-Cell Responses in AAV1 Muscle Gene Transfer
    (MDPI, 2024-09-24) Li, Ning; Kumar, Sandeep R. P.; Cao, Di; Munoz-Melero, Maite; Arisa, Sreevani; Brian, Bridget A.; Greenwood, Calista M.; Yamada, Kentaro; Duan, Dongsheng; Herzog, Roland W.; Pediatrics, School of Medicine
    While adeno-associated viral (AAV) vectors are successfully used in a variety of in vivo gene therapy applications, they continue to be hampered by the immune system. Here, we sought to identify innate and cytokine signaling pathways that promote CD8+ T-cell responses against the transgene product upon AAV1 vector administration to murine skeletal muscle. Eliminating just one of several pathways (including DNA sensing via TLR9, IL-1 receptor signaling, and possibly endosomal sensing of double-stranded RNA) substantially reduced the CD8+ T-cell response at lower vector doses but was surprisingly ineffective at higher doses. Using genetic, antibody-mediated, and vector engineering approaches, we show that blockade of at least two innate pathways is required to achieve an effect at higher vector doses. Concurrent blockade of IL-1R1 > MyD88 and TLR9 > MyD88 > type I IFN > IFNaR pathways was often but not always synergistic and had limited utility in preventing antibody formation against the transgene product. Further, even low-frequency CD8+ T-cell responses could eliminate transgene expression, even in MyD88- or IL-1R1-deficient animals that received a low vector dose. However, we provide evidence that CpG depletion of vector genomes and including TLR9 inhibitory sequences can synergize. When this construct was combined with the use of a muscle-specific promoter, transgene expression in muscle was sustained with minimal local or systemic CD8+ T-cell response. Thus, innate immune avoidance/blockade strategies by themselves, albeit helpful, may not be sufficient to prevent destructive cellular responses in muscle gene transfer because of the redundancy of immune-activating pathways.
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    SERCA2a overexpression improves muscle function in a canine Duchenne muscular dystrophy model
    (Elsevier, 2024-05-20) Kodippili, Kasun; Hakim, Chady H.; Burke, Matthew J.; Yue, Yongping; Teixeira, James A.; Zhang, Keqing; Yao, Gang; Babu, Gopal J.; Herzog, Roland W.; Duan, Dongsheng; Pediatrics, School of Medicine
    Excessive cytosolic calcium accumulation contributes to muscle degeneration in Duchenne muscular dystrophy (DMD). Sarco/endoplasmic reticulum calcium ATPase (SERCA) is a sarcoplasmic reticulum (SR) calcium pump that actively transports calcium from the cytosol into the SR. We previously showed that adeno-associated virus (AAV)-mediated SERCA2a therapy reduced cytosolic calcium overload and improved muscle and heart function in the murine DMD model. Here, we tested whether AAV SERCA2a therapy could ameliorate muscle disease in the canine DMD model. 7.83 × 1013 vector genome particles of the AAV vector were injected into the extensor carpi ulnaris (ECU) muscles of four juvenile affected dogs. Contralateral ECU muscles received excipient. Three months later, we observed widespread transgene expression and significantly increased SERCA2a levels in the AAV-injected muscles. Treatment improved SR calcium uptake, significantly reduced calpain activity, significantly improved contractile kinetics, and significantly enhanced resistance to eccentric contraction-induced force loss. Nonetheless, muscle histology was not improved. To evaluate the safety of AAV SERCA2a therapy, we delivered the vector to the ECU muscle of adult normal dogs. We achieved strong transgene expression without altering muscle histology and function. Our results suggest that AAV SERCA2a therapy has the potential to improve muscle performance in a dystrophic large mammal.