Proinflammatory cytokines have been approved by the Food and Drug Administration for the treatment of metastatic melanoma and renal carcinoma. However, effective cytokine therapy requires high-dose infusions that can result in antidrug antibodies and/or systemic side effects that limit long-term benefits. To overcome these limitations, we developed a clinically translatable cytokine delivery platform composed of polymer-encapsulated human ARPE-19 (RPE) cells that produce natural cytokines. Tumor-adjacent administration of these capsules demonstrated predictable dose modulation with spatial and temporal control and enabled peritoneal cancer immunotherapy without systemic toxicities. Interleukin-2 (IL2)–producing cytokine factory treatment eradicated peritoneal tumors in ovarian and colorectal mouse models. Furthermore, computational pharmacokinetic modeling predicts clinical translatability to humans. Notably, this platform elicited T cell responses in NHPs, consistent with reported biomarkers of treatment efficacy without toxicity. Combined, our findings demonstrate the safety and efficacy of IL2 cytokine factories in preclinical animal models and provide rationale for future clinical testing in humans.

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Adeno-associated virus (AAV) vector-based gene therapies can be applied to a wide range of diseases. AAV expression can last for months to years, but vector re-administration may be necessary to achieve life-long treatment. Unfortunately, immune system response against these vectors is potentiated after the first administration, which prevents the clinical use of repeated administration of AAVs. Reducing immune response against AAVs while minimizing immunosuppression would improve gene delivery efficiency and long-term safety. In this study, we quantified the contributions of multiple immune system components towards AAV response in mice. We identified B-cell-mediated immunity as a critical component preventing vector readministration. Specifically, we found that IgG depletion was insufficient to enhance readministration, suggesting the key role of B-cell mediated IgM antibodies in the immune response against AAV. Further, we also found that AAV-mediated transduction is improved compared to wild-type mice in µMT mice that lack functional IgM heavy chains and cannot form mature Bcells. Combined, our results suggest that IgM production in B cells is a potential target for therapeutics enabling AAV re-administration. Our results also suggest that the µMT mice are a potentially useful experimental model for gene delivery studies since they allow for up to 15-fold more efficient gene delivery.

Focused Ultrasound Blood-Brain Barrier Opening (FUS-BBBO) can deliver adeno-associated viral vectors (AAVs) to treat genetic disorders of the brain. However, such disorders often affect large brain regions. Moreover, the applicability of FUS-BBBO in the treatment of brain-wide genetic disorders has not yet been evaluated. Herein, we evaluated the transduction efficiency and safety of opening up to 105 sites simultaneously. Increasing the number of targeted sites increased gene delivery efficiency at each site. We achieved transduction of up to 60% of brain cells with comparable efficiency in the majority of the brain regions. Furthermore, gene delivery with FUS-BBBO was safe even when all 105 sites were targeted simultaneously without negative effects on animal weight, neuronal loss, or astrocyte activation. To evaluate the application of multi-site FUS-BBBO for gene therapy, we used it for gene editing using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system. FUS-BBBO-based gene delivery also leads to a significant loss of neurons at the targeted sites. Overall, this study provides a brain-wide map of transduction efficiency and the first example of multi-site gene editing after noninvasive gene delivery with FUS-BBBO.

Adeno-associated virus (AAV) vector-based gene therapies can be applied to a wide range of diseases. AAV expression can last for months to years, but vector re-administration may be necessary to achieve life-long treatment. Unfortunately, immune system response against these vectors is potentiated after the first administration, which prevents the clinical use of repeated administration of AAVs. Reducing immune response against AAVs while minimizing immunosuppression would improve gene delivery efficiency and long-term safety. In this study, we quantified the contributions of multiple immune system components towards AAV response in mice. We identified B-cell-mediated immunity as a critical component preventing vector re-administration. Specifically, we found that IgG depletion was insufficient to enhance re-administration, suggesting the key role of B-cell mediated IgM antibodies in the immune response against AAV. Further, we also found that AAV-mediated transduction is improved compared to wild-type mice in µMT mice that lack functional IgM heavy chains and cannot form mature B-cells. Combined, our results suggest that IgM production in B cells is a potential target for therapeutics enabling AAV re-administration. Our results also suggest that the µMT mice are a potentially useful experimental model for gene delivery studies since they allow for up to 15-fold more efficient gene delivery.