Molecular Therapy-Methods & Clinical Development最新文献

Pompe disease is a glycogen storage disorder caused by mutations in the acid α-glucosidase (GAA) gene, leading to reduced GAA activity and glycogen accumulation in heart and skeletal muscles. Enzyme replacement therapy with recombinant GAA, the standard of care for Pompe disease, is limited by poor skeletal muscle distribution and immune responses after repeated administrations. The expression of GAA in muscle with adeno-associated virus (AAV) vectors has shown limitations, mainly the low targeting efficiency and immune responses to the transgene. To address these issues, we developed AAV capsids with improved skeletal muscle targeting and reduced off-targeting. These capsids combined with codon optimization, muscle-specific cis-regulatory elements, and a synthetic promoter demonstrated a strong skeletal muscle tropism, reduced liver targeting, and enhanced GAA transgene expression and reduced glycogen accumulation in a Gaa ^-/- mouse model. However, increased muscle-specific expression led to a robust anti-hGAA immune response. To circumvent this, the AAVMYO2 capsid was tested in immunodeficient Gaa ^-/- Cd4 ^-/- mice and compared to AAV9 at different doses. The combination of AAVMYO2 with an optimized transgene expression cassette provided a dose-dependent advantage for glycogen reduction in skeletal muscles of Gaa ^-/- Cd4 ^-/- mice. These findings support the potential of muscle-specific AAV gene therapy for Pompe disease at lower doses with greater specificity.

Lipid nanoparticles (LNPs) are lead non-viral vectors for delivering nucleic acids. LNPs can efficiently encapsulate nucleic acids, protect them from degradation, enhance cellular uptake and induce endosome escape, which show high transfection efficiency and low immunogenicity. In this review, we first introduce the LNP components, highlighting their critical roles in encapsulation, stability, delivery efficiency, and tissue tropism. Next, we summarize different techniques for LNP formulation with a focus on their advantages and disadvantages. Then, we discuss the diverse applications of LNPs in preclinical and clinical studies. Finally, we provide perspectives in the future development of LNPs.

Bietti crystalline dystrophy (BCD) is an autosomal recessive disorder caused by loss-of-function mutations in the CYP4V2 gene, characterized by crystal-like lipid deposits in the retina, progressive photoreceptor loss, and retinal pigment epithelium (RPE) deterioration. Currently, there are no approved treatments for BCD. VGR-R01, an investigational gene therapy, uses subretinal administration of recombinant adeno-associated virus type 8 (AAV8) vector to deliver the human CYP4V2 gene. This therapy is now undergoing phase 1/2 clinical trials (NCT05694598). The pre-clinical study results for VGR-R01 are summarized, with a focus on its pharmacology, pharmacokinetics, and toxicology. The in vitro cellular studies demonstrated that VGR-R01 induces a dose-dependent expression of the CYP4V2 protein, which significantly enhances fatty acid hydroxylase activity and reduces lipid droplet accumulations in the RPE cells. In vivo, VGR-R01 showed effectiveness in improving electroretinogram (ERG) amplitudes in 8-month-old Cyp4v3 ^-/- mice. VGR-R01 was well tolerated in New Zealand rabbits and non-human primates (NHPs). Furthermore, after subretinal administration, VGR-R01 was primarily distributed in the ocular tissues, especially in the retina, with minimal systemic presence, notably in the gonads. Overall, these results support the potential for clinical application of VGR-R01 in treating BCD.

Noise-induced hearing loss (NIHL) poses an emerging global health problem with only ear protection or sound avoidance as preventive strategies. The cochlea receives some protection from medial olivocochlear efferent neurons, providing a potential target for therapeutic enhancement. Cholinergic efferents release acetylcholine (ACh) to hyperpolarize and shunt the outer hair cells (OHCs), reducing sound-evoked activation. The (α9)₂(α10)₃ nicotinic ACh receptor (nAChR) on the OHCs mediates this effect. Transgenic knockin mice with a gain-of-function nAChR (α9L9'T) suffer less NIHL. α9 knockout mice are more vulnerable to NIHL but can be rescued by viral transduction of the α9L9'T subunit. In this study, an HA-tagged gain-of-function α9 isoform was expressed in wild-type mice to reduce NIHL. Synaptic integration of the virally expressed nAChR subunit was confirmed by HA immunopuncta localized to the postsynaptic membrane of OHCs. After noise exposure, AAV2.7m8-CAG-α9L9'T-HA (α9L9'T-HA)-injected mice had less hearing loss (auditory brainstem response [ABR] thresholds and threshold shifts) than did control mice. ABRs of α9L9'T-HA-injected mice also had larger wave-1 amplitudes and better recovery of wave-1 amplitudes post noise exposure. Thus, virally expressed α9L9'T combines effectively with native α9 and α10 subunits to mitigate NIHL in wild-type cochleas.

We developed an in vivo hematopoietic stem cell (HSC) gene therapy approach consisting of HSC mobilization and intravenous injection of helper dependent adenovirus (HDAd) vectors. While we have demonstrated safety and efficacy of the in vivo approach in CD46-transgenic mice and rhesus macaques, studies in mice with a humanized hematopoietic system could facilitate its potential clinical translation for the treatment of hemoglobinopathies and HIV. Using mild myelo-conditioning in NSGW41 mice and cryopreserved human CD34⁺ cells from healthy donors we achieved ∼10% human chimerism in peripheral blood. Engrafted primitive human CD45⁺/CD34⁺/CD90⁺-HSCs efficiently mobilized by different approaches involving AMD3100 in combination with granulocyte colony-stimulating factor G-CSF, truncated Groβ (tGroβ), or WU106/tGroβ. At the peak of mobilization, integrating HDAd-GFP vectors were injected intravenously followed by O⁶BG/BCNU in vivo selection. Long-term stable GFP expression was shown for HDAd5/35 and the new vector platforms HDAd6/3 and HDAd5/35_lam, a fiber/penton-modified vector. Two months post transduction, GFP marking in the periphery were 22.38% (8.17%), 41.12% (10.62%), and 32.15% (4.49%) for HDAd5/35, HDAd6/3, and HDAd5/35_lam, respectively. GFP levels in bone marrow were 33.53% (8.96%), 53.51% (6.95%), and 33.29% (5.21%) and in spleen 32.6% (9.25%), 33.75% (5.47%), and 20.79% (6.15%). Our study describes a new animal model for in vivo HSC transduction with HDAds, with implications for studies with other vectors.

In vivo gene therapy targeting hematopoietic stem cells (HSCs) holds significant therapeutic potential for treating hematological diseases. This study uses adeno-associated virus serotype 6 (AAV6) vectors and Cre recombination to systematically optimize the parameters for effective in vivo HSC transduction. We evaluated various genetic architectures and delivery methods of AAV6, establishing an optimized protocol that achieved functional recombination in more than two-thirds of immunophenotypic HSCs. Our findings highlight that second-strand synthesis is a critical limiting factor for transgene expression in HSCs, leading to significant under-detection of HSC transduction with single-stranded AAV6 vectors. We also demonstrate that HSCs in the bone marrow (BM) are readily accessible to transduction, with neither localized injection nor mobilization of HSCs into the bloodstream, enhancing transduction efficacy. Additionally, we observed a surprising preference for HSC transduction over other BM cells, regardless of the AAV6 delivery route. Together, these findings not only underscore the potential of AAV vectors for in vivo HSC gene therapy but also lay a foundation that can inform the development of both in vivo AAV-based HSC gene therapies and potentially in vivo HSC gene therapies that employ alternative delivery modalities.

CRISPR-Cas9 ribonucleoproteins (RNPs) combined with a nucleic acid template encoding a chimeric antigen receptor (CAR) transgene can edit human cells to produce CAR T cells with precise CAR insertion at a single locus. However, many human cells have adverse innate immune responses to foreign nucleic acids, particularly circular double-stranded DNA (dsDNA). Here, we introduce Cleaved, LInearized with Protein Template (Cas9-CLIPT), a circular plasmid containing a single target sequence for the Cas9 RNP, such that during manufacturing, Cas9-RNP binds and cleaves the plasmid to linearize the dsDNA in vitro. Cas9-RNP remains bound to the linearized template and is delivered to cells to promote precise knock-in via homology-directed repair with Cas9-CLIPT. Cas9-CLIPT Nanoplasmids generate up to 1.7-fold higher rates of precise knock-in relative to linearized dsDNA, reaching efficiencies up to 60% with non-homologous end joining inhibition. Cas9-CLIPT-manufactured GD2 TRAC-CAR T cells are potent against GD2⁺ neuroblastoma cells and exhibit an enriched stem cell memory phenotype. On several electroporation instruments and approaching clinically relevant yields, we successfully manufactured TRAC-CAR T cells using Cas9-CLIPT plasmids containing large (2-6 kb) transgenes. Cas9-CLIPT strategies have the potential to simplify donor template production and integrate large transgenes, allowing for more efficient nonviral manufacturing of multifunctional, genome-edited immune cell therapies.

Lipid nanoparticles (LNPs) are now highly effective transporters of nucleic acids to the liver. This liver-specificity is largely due to their association with certain serum proteins, most notably apolipoprotein E (ApoE), which directs them to liver cells by binding to the low-density lipoprotein (LDL) receptors on hepatocytes. The liver's distinct anatomy, with its various specialized cell types, also influences how LNPs are taken up from the circulation, cleared, and how effective they are in delivering treatments. In this review, we consider factors that facilitate LNP's effective liver targeting and explore the latest advances in liver-targeted LNP technologies. Understanding how LNPs are targeted to the liver can help for effective design and optimization of nanoparticle-based therapies. Comprehension of the cellular interaction and biodistribution of LNPs not only leads to better treatments for liver diseases but also delivers insight for directing nanoparticles to other tissues, potentially broadening their range of therapeutic applications.

Wilson's disease (WD) is an autosomal recessive disorder caused by pathogenic variants in the ATP7B gene, resulting in the toxic accumulation of copper (Cu). Impaired Cu homeostasis in WD is characterized by low serum ceruloplasmin, excess hepatic Cu, and elevated urinary Cu. WD often presents with hepatic and/or neurological diseases and is fatal if untreated. Adeno-associated virus (AAV)-mediated gene therapy holds promise for WD, but challenges remain in efficacy and safety. Here, we established an Atp7b R780L knockin (KI) mouse model corresponding to the human ATP7B R778L variant and investigated the therapeutic efficacy and safety of liver-targeted AAV8-mediated ATP7B (AAV8-ΔC4ATP7B) gene therapy in this model. The results demonstrated the Atp7b ^KI/KI mice recapitulated key features of impaired Cu metabolism in WD but had mild liver disease. Ten-week-old Atp7b ^KI/KI mice received a single-dose of AAV8-ΔC4ATP7B and were sacrificed at 8 or 30 weeks after treatment. Treated Atp7b ^KI/KI mice showed normalization of serum ceruloplasmin, reduced hepatic Cu, decreased urinary Cu, and reversed liver histopathology. Serum transaminases had a transient increase at 8 weeks after treatment but returned to normal at 30 weeks after treatment. These data provide evidence for the efficacy and safety of AAV8-ΔC4ATP7B in animals, supporting clinical translation to patients with WD.

Recombinant adeno-associated viruses (AAVs) have been widely used for gene delivery and gene therapy. However, certain AAV serotypes exhibited distinct transduction patterns among different mouse strains or between mice and non-human primates (NHPs). These variations prompted us to investigate the AAV tropism of 21 capsid variants using barcoded AAV libraries among different tissues in C57BL/6 and BALB/c mice, as well as in crab-eating macaques. Our study unveiled that AAV tropisms varied significantly among different mouse strains and species, particularly in capsid variants such as AAV4, AAV9, PHP.B, and CAP-B10. Notably, AAV4 exhibited liver-detargeting properties in both mice and NHPs, and was remarkably efficient in transducing the lung, glomerulus, and pancreatic islet. These findings furnish crucial insights into the variations of AAV tropism among different mouse strains and species and facilitate the selection of appropriate AAV capsids for target tissues among different mouse strains and in NHPs.