Summary X-Linked severe combined immunodeficiency (SCID-X1) is a genetic disease that leaves newborns at high risk of serious infection and a predicted lifespan of less than one year in the absence of a matched bone marrow donor. The disease pathogenesis is due to mutations in the gene encoding the Interleukin-2 receptor gamma chain (IL-2Rγ) leading to a lack of functional lymphocytes. With the leukemogenic concerns of viral gene therapy there is a need to explore alternative therapeutic options. We have utilized induced pluripotent stem cell (iPSC) technology and genome editing mediated by TALENs to generate isogenic patient-specific mutant and gene corrected iPSC lines. While the patient-derived mutant iPSC have the capacity to generate hematopoietic precursors and myeloid cells, only wild-type and gene-corrected iPSC can additionally generate mature NK-cells and T-cell precursors expressing the correctly spliced IL-2Rγ. This study highlights the potential for the development of autologous cell therapy for SCID-X1 patients.

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Adeno-associated viruses (AAV) rely on helper viruses to transition from latency to lytic infection. Some AAV serotypes are secreted in a pre-lytic manner as free or extracellular vesicle (EV)-associated particles, although mechanisms underlying such are unknown. Here, we discover that the membrane-associated accessory protein (MAAP), expressed from a frameshifted open reading frame in the AAV cap gene, is a novel viral egress factor. MAAP contains a highly conserved, cationic amphipathic domain critical for AAV secretion. Wild type or recombinant AAV with a mutated MAAP start site (MAAPΔ) show markedly attenuated secretion and correspondingly, increased intracellular retention. Trans-complementation with MAAP restored secretion of multiple AAV/MAAPΔ serotypes. Further, multiple processing and analytical methods corroborate that one plausible mechanism by which MAAP promotes viral egress is through AAV/EV association. In addition to characterizing a novel viral egress factor, we highlight a prospective engineering platform to modulate secretion of AAV vectors or other EV-associated cargo.

The immune response to Staphylococcus aureus infection in skin involves the recruitment of neutrophils (PMN) from the bone marrow via the circulation and local granulopoiesis from hematopoietic stem and progenitor cells (HSPC) that also traffic to infected skin wounds. We focus on regulation of PMN number and function and the role of pore-forming alpha-toxin (AT), a virulence factor that causes host cell lysis and elicits inflammasome-mediated IL-1β secretion in wounds. Infection with wild type S. aureus enriched in AT reduced PMN recruitment and resulted in sustained bacterial burden and delayed wound healing. In contrast, PMN recruitment to wounds infected with an isogenic AT mutant strain (ΔAT) was unimpeded, exhibiting efficient bacterial clearance and hastened wound resolution. HSPC recruited to infected wounds was unaffected by AT production and were activated to expand PMN numbers in proportion to S. aureus abundance in a manner regulated by TLR2 and IL-1 receptor signaling. Immunodeficient MyD88 knockout mice infected with S. aureus experienced lethal sepsis that was reversed by PMN expansion mediated by injection of wild type HSPC directly into wounds. We conclude that AT induced IL-1β promotes local granulopoiesis and effective resolution of S. aureus-infected wounds, revealing a potential antibiotic free strategy for tuning the innate immune response to treat MRSA infection in immunodeficient patients.