Gene regulation in inborn errors of immunity: Implications for gene therapy design and efficacy.

Inborn errors of immunity (IEI) present a unique paradigm in the realm of gene therapy, emphasizing the need for precision in therapeutic design. As gene therapy transitions from broad-spectrum gene addition to careful modification of specific genes, the enduring safety and effectiveness of these therapies in clinical settings have become crucial. This review discusses the significance of IEIs as foundational models for pioneering and refining precision medicine. We explore the capabilities of gene addition and gene correction platforms in modifying the DNA sequence of primary cells tailored for IEIs. The review uses four specific IEIs to highlight key issues in gene therapy strategies: X-linked agammaglobulinemia (XLA), X-linked chronic granulomatous disease (X-CGD), X-linked hyper IgM syndrome (XHIGM), and immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX). We detail the regulatory intricacies and therapeutic innovations for each disorder, incorporating insights from relevant clinical trials. For most IEIs, regulated expression is a vital aspect of the underlying biology, and we discuss the importance of endogenous regulation in developing gene therapy strategies.

Combining Cell-Intrinsic and -Extrinsic Resistance to HIV-1 By Engineering Hematopoietic Stem Cells for CCR5 Knockout and B Cell Secretion of Therapeutic Antibodies.

Autologous transplantation of CCR5 null hematopoietic stem and progenitor cells (HSPCs) is the only known cure for HIV-1 infection. However, this treatment is limited because of the rarity of CCR5 -null matched donors, the morbidities associated with allogeneic transplantation, and the prevalence of HIV-1 strains resistant to CCR5 knockout (KO) alone. Here, we propose a one-time therapy through autologous transplantation of HSPCs genetically engineered ex vivo to produce both CCR5 KO cells and long-term secretion of potent HIV-1 inhibiting antibodies from B cell progeny. CRISPR-Cas9-engineered HSPCs maintain engraftment capacity and multi-lineage potential in vivo and can be engineered to express multiple antibodies simultaneously. Human B cells engineered to express each antibody secrete neutralizing concentrations capable of inhibiting HIV-1 pseudovirus infection in vitro . This work lays the groundwork for a potential one-time functional cure for HIV-1 through combining the long-term delivery of therapeutic antibodies against HIV-1 and the known efficacy of CCR5 KO HSPC transplantation.

Genetically corrected RAG2-SCID human hematopoietic stem cells restore V(D)J-recombinase and rescue lymphoid deficiency.

ABSTRACT: Recombination-activating genes (RAG1 and RAG2) are critical for lymphoid cell development and function by initiating the variable (V), diversity (D), and joining (J) (V(D)J)-recombination process to generate polyclonal lymphocytes with broad antigen specificity. The clinical manifestations of defective RAG1/2 genes range from immune dysregulation to severe combined immunodeficiencies (SCIDs), causing life-threatening infections and death early in life without hematopoietic cell transplantation (HCT). Despite improvements, haploidentical HCT without myeloablative conditioning carries a high risk of graft failure and incomplete immune reconstitution. The RAG complex is only expressed during the G0-G1 phase of the cell cycle in the early stages of T- and B-cell development, underscoring that a direct gene correction might capture the precise temporal expression of the endogenous gene. Here, we report a feasibility study using the CRISPR/Cas9-based "universal gene-correction" approach for the RAG2 locus in human hematopoietic stem/progenitor cells (HSPCs) from healthy donors and RAG2-SCID patient. V(D)J-recombinase activity was restored after gene correction of RAG2-SCID-derived HSPCs, resulting in the development of T-cell receptor (TCR) αß and γδ CD3+ cells and single-positive CD4+ and CD8+ lymphocytes. TCR repertoire analysis indicated a normal distribution of CDR3 length and preserved usage of the distal TRAV genes. We confirmed the in vivo rescue of B-cell development with normal immunoglobulin M surface expression and a significant decrease in CD56bright natural killer cells. Together, we provide specificity, toxicity, and efficacy data supporting the development of a gene-correction therapy to benefit RAG2-deficient patients.

Allele-Specific Gene Editing Rescues Pathology in a Human Model of Charcot-Marie-Tooth Disease Type 2E.

Many neuromuscular disorders are caused by dominant missense mutations that lead to dominant-negative or gain-of-function pathology. This category of disease is challenging to address via drug treatment or gene augmentation therapy because these strategies may not eliminate the effects of the mutant protein or RNA. Thus, effective treatments are severely lacking for these dominant diseases, which often cause severe disability or death. The targeted inactivation of dominant disease alleles by gene editing is a promising approach with the potential to completely remove the cause of pathology with a single treatment. Here, we demonstrate that allele-specific CRISPR gene editing in a human model of axonal Charcot-Marie-Tooth (CMT) disease rescues pathology caused by a dominant missense mutation in the neurofilament light chain gene (NEFL, CMT type 2E). We utilized a rapid and efficient method for generating spinal motor neurons from human induced pluripotent stem cells (iPSCs) derived from a patient with CMT2E. Diseased motor neurons recapitulated known pathologic phenotypes at early time points of differentiation, including aberrant accumulation of neurofilament light chain protein in neuronal cell bodies. We selectively inactivated the disease NEFL allele in patient iPSCs using Cas9 enzymes to introduce a frameshift at the pathogenic N98S mutation. Motor neurons carrying this allele-specific frameshift demonstrated an amelioration of the disease phenotype comparable to that seen in an isogenic control with precise correction of the mutation. Our results validate allele-specific gene editing as a therapeutic approach for CMT2E and as a promising strategy to silence dominant mutations in any gene for which heterozygous loss-of-function is well tolerated. This highlights the potential for gene editing as a therapy for currently untreatable dominant neurologic diseases.