Molecular Therapy-Methods & Clinical Development最新文献

Autologous transplantation of ex vivo gene-modified/corrected hematopoietic stem cells (HSCs) offers a definitive therapeutic approach to restore hematopoiesis in Fanconi anemia (FA) patients. However, this approach not only requires ex vivo treatment of patient HSCs in specialized facilities but also inevitably results in a loss of fragile and limited patient HSCs. In vivo correction of HSCs directly in the patient can overcome these limitations. To develop such in vivo gene therapy (GT) strategies, an appropriate in vivo model with sufficient target human HSCs carrying the disease-associated mutation is crucial. However, due to the proliferative defect imposed on FA-mutant cells, it is difficult to establish a humanized mouse model with a high engraftment of mutant HSCs. Here, we report a humanized mouse model of FA that results in high chimerism with FA-mutant human HSCs. We demonstrate successful engraftment, uncompromised proliferation, and long-term persistence of the FA-mutant HSCs facilitated by the full-length FANCA expression introduced via a lentiviral vector. This model resolves the lack of an in vivo FA disease model with human HSCs and is a promising platform for testing in vivo gene editing strategies targeting human cells.

Cardiovascular diseases, especially atherosclerosis, are the main cause of death in the whole world. The risk can be reduced by lowering the serum low-density lipoprotein cholesterol by targeting proprotein convertase 9 (PCSK9) through genome editing or neutralization by monoclonal antibodies. Vaccination against PCSK9 represents an alternative with potentially long-lasting efficacy, but must overcome the challenge of immunogenicity against the endogenous protein, which can also elicit lower antibody response due to B cell tolerance. In contrast to the previously reported weakly immunogenic PCSK9 peptides, we have developed a designed chimeric PCSK9 that maintains the surface epitopes and elicits a B cell immune response with PCSK9-specific antibodies, comparable to human-based vaccines, but eliminates the natural protein T cell epitopes and impairs self-antigen-mediated T cell cytotoxicity upon vaccination. We demonstrated that vaccination with chimera-based vaccines generates humoral immunity with a decreased T cell reactivity. In an atherosclerosis mouse model, the effect persisted over 20 weeks, as evidenced by a reduction in the circulating PCSK9 and cholesterol and a lower atherosclerotic disease burden in the aorta. This demonstrates a therapeutic improvement in atherosclerosis in an animal model and the proof-of-concept for the rational design of vaccines against endogenous proteins.

Gene replacement therapy is becoming a therapeutic option for patients with Duchenne muscular dystrophy. Truncated dystrophins that can be expressed from adeno-associated viral (AAV) vectors have been designed and shown to retain many, though not all, functional features of the full-length protein. These "microdystrophins" differ by their combination of hinges and spectrin-like repeats and the inclusion of functional domains. Several microdystrophins have advanced to the clinic, and although all have been shown to restore muscle force in preclinical models, they may differ in the efficiency of providing muscle resilience and resistance to contraction-induced stress. Here, we examine whether the inclusion of a highly evolutionarily conserved domain found at the C-Terminus of dystrophin may improve the therapeutic activity of microdystrophin. We find that adding this portion of the C-Terminal domain results in a microdystrophin that is maintained at higher levels in the transduced muscles, recruits the dystrophin-associated protein complex more effectively to the sarcolemma, provides improved histopathological benefit, and increases muscle force and resistance to damage in mice lacking dystrophin. These findings indicate that incorporation of the dystrophin C-Terminus is an enhancement for microdystrophin design and may improve functional benefit.

Facioscapulohumeral muscular dystrophy (FSHD) is the third most diagnosed muscular dystrophy. The disease is caused by genetic and epigenetic disruptions that result in misexpression of the germline transcription factor DUX4 in skeletal muscle, leading to muscle toxicity and turnover. As a gene misexpressed exclusively in muscle, DUX4 is a suitable for muscle-targeted small interfering RNA (siRNA) knockdown therapy. Here we identify a DUX4-targeting siRNA, DU01, that potently knocks down the expression of DUX4 target genes in FSHD patient-derived myotubes ex vivo. Further, DU01 conjugated with the lipid docosanoic acid (DCA) is systemically deliverable to mice by subcutaneous injection to achieve greater than 50% knockdown of DUX4 target genes in FSHD patient muscle xenografts. These findings identify the DCA-conjugated DUX4 siRNA, DCA-siRNA^DUX4, as a disease-targeting therapeutic for clinical development.

Multiple sclerosis (MS) is a neurological disease characterized by demyelinating lesions in the CNS. This study investigated whether a minimally invasive adeno-associated virus (AAV) vector (AAV.PHP.eB) can direct transgene expression in CNS cell types relevant to MS, including astrocytes, oligodendrocytes, oligodendrocyte precursor cells (OPCs), microglia, and neurons in experimental autoimmune encephalitis, a widely used MS model. In vivo bioluminescence imaging and histological analysis following AAV.PHP.eB-mediated gene delivery in healthy mice using the ubiquitous CAG promoter and five neural promoters (MBP, Sox10, hSyn1, gfa2, and gfaABC1D) revealed long-term, robust, and cell-type-specific activity across the brain and spinal cord. AAV.PHP.eB is capable of traversing the blood-brain barrier in experimental autoimmune encephalitis (EAE) and directs sustained and cell-type-specific transgene expression for the MBP, Sox10, hSyn1, and gfaABC1D promoters. The MBP and Sox10 promoters directed transgene expression in oligodendroglia around and within inflammatory demyelinating lesions, whereas the gfaABC1D promoter directs transgene expression in gray and white matter astrocytes and hSyn1 in neurons. The neural promoters were minimally active in the periphery, with the exception of gfa2. This methodological study is a first step toward the development of minimally invasive gene therapy to promote myelin repair and/or suppress inflammation in MS.

In vivo genome editing with CRISPR-Cas9 systems is generating worldwide attention and enthusiasm for the possible treatment of genetic disorders. However, the consequences of potential immunogenicity of the bacterial Cas9 protein and the AAV capsid have been the subject of considerable debate. Here, we model the antigen presentation in cells after in vivo gene editing by in vitro transduction of a human cell line with an AAV2 vector that delivers the Staphylococcus aureus Cas9 transgene. Through HLA class I enrichment, peptide elution, and highly sensitive LC-MS interrogation, we identified a highly conserved saCas9-derived T cell epitope in the catalytic domain of the enzyme that is restricted to HLA-A^∗02:01 and induces CD8+ T cell activation and killing. We conclude that AAV delivery of Cas9 results in presentation of a T cell epitope that can activate CD8+ cells and induce killing of the transduced cell, with important ramifications for in vivo genome editing strategies.

Safe and efficient nucleic acid delivery to targeted cell populations remains a challenge in the fields of cell and gene therapy. Toward this end, we attempted to utilize the "DogTag-DogCatcher" system to target adenoviral vectors. "DogTag" is a short peptide that forms a spontaneous isopeptide bond upon mixing with its partner protein, "DogCatcher." We genetically incorporated the DogTag peptide into the protein responsible for initial binding of the virus to its target cell, the fiber. This allowed permanent linking of DogCatcher-fused single-domain or single-chain antibodies at the fiber. This modification allowed simple, effective, and exclusive targeting of the vector to cells bound by the linked antibody. This enhanced gene transfer into primary B and T cells by up to 60-fold in vitro and 2- to 3-fold in vivo in mice without other alterations to vector tropism. Although the system's in vivo performance is currently suboptimal and additional engineering is needed prior to further use, these studies form the basis of a novel method for targeting adenovirus that can be combined with additional well-characterized adenovirus modifications toward applications in cell engineering, gene therapy, vaccines, oncolytics, and others.

The ability to deliver a therapeutic sequence to a specific cell type in the human brain would make possible innumerable therapeutic options for some of our most challenging diseases; however, studies on adeno-associated virus (AAV) vector tropism have generally relied on animal models with limited translational utility. For this reason, establishing the tropism of common adeno-associated virus (AAV) vectors in living human brain tissue serves as an important baseline for further optimization, as well as a determination of human brain cell types transduced by clinically approved gene therapy vectors AAV2 and AAV9. We have adapted an ex vivo organotypic model to evaluate AAV transduction properties in living slices of human brain tissue. Using fluorescent reporter expression and single-nucleus RNA sequencing, we found that common AAV vectors show broad transduction of normal cell types, with protein expression most apparent in astrocytes; this work introduces a pipeline for identifying and optimizing AAV gene therapy vectors in human brain samples.

Saponins are a class of phytocompounds known for their amphiphilic properties. Here, we have evaluated incorporation of 40 saponins into a model lipid nanoparticle (LNP) formulation and evaluated their performance in vitro and in vivo. We reasoned that the surfactant activity of saponins could be beneficial in the context of cell and gene therapy due to the disruption of the intracellular membranes. We established formulation methodology to incorporate saponins into LNPs and measured their endosomal disruption and transfection efficiency with DNA barcode and mRNA cargoes. We identified two saponins-quillaic acid and macranthoidin B-that increase the LNP transfection efficiency and endosomal disruption. Saponin formulations demonstrated cargo-dependent activation of the innate immune system, as measured by the cell-based assays of interferon regulatory factor (IRF) and NF-κB pathway activation. Quillaic acid LNPs resulted in higher titers of anti-OVA IgG2a in the vaccination studies compared to a "naive" LNP control, which suggests a more Th1-biased immunopathology of these vaccines. As Th2-biased vaccines can trigger an allergic response, an mRNA vaccine with a balanced Th1/Th2 response is more favorable for translation into the clinic. Overall, quillaic acid may serve as an adjuvant for mRNA vaccines and potentially decrease the risk of vaccine-associated adverse events.