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Adrenomyeloneuropathy is a progressive neurodegenerative disease caused by pathogenic variants in the ABCD1 gene, resulting in very-long-chain fatty acid (VLCFA) accumulation that leads to dying-back axonopathy. Our candidate gene therapy, SBT101 (AAV9-human ABCD1 [h ABCD1 ]), aims to ameliorate pathology by delivering functional copies of h ABCD1 to the spinal cord. Transduced cells produce functional ABCD1 protein, thereby repairing the underlying biochemical defect. In vitro and in vivo mouse studies were conducted to assess the biochemical and functional efficacy of SBT101 and show effective delivery to target tissues involved in the disease pathology: spinal cord and dorsal root ganglia. Administration of SBT101 to mixed glial cell cultures from Abcd1 -Null mice, and to male Abcd1 knockout ( Abcd1 -/y ) and double-knockout ( Abcd1 -/y / Abcd2 -/- ) mice led to increased hABCD1 production and reduced VLCFA. Double-knockout mice also exhibited improved grip strength. Furthermore, we conducted biodistribution and safety assessments in nonhuman primates. Six-hour intrathecal lumbar infusions demonstrated effective transduction throughout target tissues, supporting the clinical feasibility of the procedure. SBT101 was well tolerated, with no observed SBT101-related mortality or clinical signs. These findings not only provide preclinical efficacy data for SBT101 but also inform clinically relevant SBT101 dose selection for patients with adrenomyeloneuropathy., Competing Interests: T.L. and K.K. are employees and stock/shareholders of SwanBio Therapeutics. V.V., D.W.A., S.W.C., and H.P. were employees and stock/shareholders of SwanBio Therapeutics at the time the studies were performed: V.V. is an employee of Code Biotherapeutics; D.W.A. is an employee of Code Biotherapeutics; S.W.C. is an employee of Clithero-Clark Consulting, LLC; H.P. is an employee of Center for Breakthrough Medicines. C.A.M. has financial interests in/received consultancy fees from Chameleon Biosciences, Skylark Bio, and Sphere Gene Therapeutics; resequenceceived research support/grants from BridgeBio, SwanBio Therapeutics, and Waypoint Capital; and received royalties for licensing agreements from BridgeBio, Partners Healthcare, Skylark Bio, Sphere Gene Therapeutics, and SwanBio Therapeutics. Y.G. received royalties for licensing agreements from SwanBio Therapeutics. F.E. received research support/grants from Aspa Therapeutics, bluebird bio, Ionis Pharmaceuticals, Minoryx Therapeutics, and Sio Gene Therapies; received consultancy fees from Autobahn Therapeutics, Poxel, SwanBio Therapeutics, Takeda, Taysha, and UpToDate; and is a founder and stock/shareholder of SwanBio Therapeutics. A.P., A.B., and S.K. received unrestricted research support/grants and consultancy fees from SwanBio Therapeutics., (© 2024 The Author(s).)

Microglia are critical innate immune cells of the brain. In vivo targeting of microglia using gene-delivery systems is crucial for studying brain physiology and developing gene therapies for neurodegenerative diseases and other brain disorders such as NeuroAIDS. Historically, microglia have been extremely resistant to transduction by viral vectors, including adeno-associated virus (AAV) vectors. Recently, there has been some progress demonstrating the feasibility and potential of using AAV to transduce microglia after direct intraparenchymal vector injection. Data suggests that combining specific AAV capsids with microglia-specific gene expression cassettes to reduce neuron off-targeting will be key. However, no groups have developed AAV capsids for microglia transduction after intracerebroventricular (ICV) injection. The ICV route of administration has advantages such as increased brain biodistribution while avoiding issues related to systemic injection. Here, we performed an in vivo selection using an AAV peptide display library that enables recovery of capsids that mediate transgene expression in microglia. Using this approach, we identified a capsid, MC5, which mediated enhanced transduction of microglia after ICV injection compared to AAV9. Furthermore, MC5 enhanced both the efficiency (85%) and specificity (93%) of transduction compared to a recently described evolved AAV9 capsid for microglia targeting after direct injection into the brain parenchyma. Exploration of the use of MC5 in a mouse models of Alzheimer's disease revealed transduced microglia surrounding and within plaques. Overall, our results demonstrate that the MC5 capsid is a useful gene transfer tool to target microglia in vivo by direct and ICV routes of administration., Competing Interests: Competing interests: C.A.M. has a financial interest in Sphere Gene Therapeutics, Inc., Chameleon Biosciences, Inc., and Skylark Bio, Inc., companies developing gene therapy platforms. C.A.M.’s interests were reviewed and are managed by MGH and Mass General Brigham in accordance with their conflict-of-interest policies. C.A.M., M.C.S., K.S.H., and P.E. have filed a patent application with claims involving the MC5 capsid.

Adeno-associated viruses (AAV) are promising vectors for gene therapy due to their efficacy in vivo. However, there is room for improvement to address key limitations such as the pre-existing immunity to AAV in patients, high-dose toxicity, and relatively low efficiency for some cell types. This study introduces a metabolic engineering approach, using knockout of the enzyme phosphatidylserine synthase 1 (PTDSS1) to increase the abundance of extracellular vesicle-enclosed AAV (EV-AAV) relative to free AAV in the supernatant of producer cells, simplifying downstream purification processes. The lipid-engineered HEK293T-ΔPTDSS1 cell line achieved a 42.7-fold enrichment of EV-AAV9 compared to free AAV9 in the supernatant. The rational genetic strategy also led to a 300-fold decrease of free AAV in supernatant compared to wild-type HEK293T. The membrane-engineered EV-AAV9 (mEV-AAV9) showed unique envelope composition alterations, including cholesterol enrichment and improved transduction efficiency in human AC16 cardiomyocytes by 1.5-fold compared to conventional EV-AAV9 and by 11-fold compared to non-enveloped AAV9. Robust in-vivo transduction four weeks after intraparenchymal administration of mEV-AAV9 was observed in the murine brain. This study shows promise in the potential of lipid metabolic engineering strategies to improve the efficiency and process development of enveloped gene delivery vectors. Keywords: membrane engineering, EV-AAV, upstream, downstream.