Human Gene Therapy

Developing gene therapies involving gene editing is a rapidly evolving field with large potential implications for improving health for both rare and common diseases. Ensuring that these technologies are developed safely, efficiently, and fairly is essential. To better understand the ethical considerations and regulatory requirements and challenges with gene therapies involving gene editing that may advance precision health. Through a multistakeholder workshop and subsequent engagements, multiple ethical and regulatory barriers to developing and implementing gene therapies involving gene editing were identified. Eight major themes emerged that warrant careful consideration, including (1) objectives (treatment, risk reduction, and enhancement) for the intervention; (2) competing interests of safety, equity, and desire for research efficiencies; (3) unique aspects of gene editing related to rare and ultrarare genetic conditions; (4) considerations in the pediatric population; (5) regulatory requirements and ethics oversight; (6) challenges with long-term follow-up and data sharing; and (7) communication. To promote the safe, efficient development of gene therapies involving gene editing that will reach their full potential, all stakeholders will have to undertake an unprecedented degree of collaboration. However, this will be essential to ensure that these interventions are effective, ethically sound, and patient-centered.

Common variants of the apolipoprotein E (APOE) gene have a major impact on the risk of developing Alzheimer’s disease (AD). Relative to homozygotes with the common E3 allele, the APOE4 variant (C112R) increases risk by 3.5-fold in E3/E4 heterozygotes and 15-fold in E4 homozygotes. Since the E3 and E4 alleles differ only by a single nucleotide, gene editing of E4 to E3 is a potential strategy to reduce AD risk in E4 homozygotes. Because the APOE pool in the brain is separate from systemic APOE, editing to treat AD would ideally be directed to the brain. Following in vitro optimization of prime editing guide RNAs, efficient prime editing expression cassettes were inserted into the adeno-associated virus (AAV) split-intein system and packaged into pairs of AAV vectors for in vivo editing. The AAV vectors were administered to human homozygous APOE4-targeted replacement mice (TRE4), and APOE4 to APOE3 editing efficiency was assessed after 4 weeks. The prime editing construct designated APOE3/4-3_10 was the most efficient at APOE4 to APOE3 conversion, both in liver following intravenous delivery and in brain following intrahippocampal delivery. To assess brain-wide editing, two AAV capsids were compared, including AAVrh.10 with administration either directly to the hippocampus or to the cerebrospinal fluid via the cisterna magna and AAV-CAP.B10 administered intravenously. Other than minor differences in APOE4/3-3_10 mediated E4 to E3 editing in the cerebellum, the different capsids and routes yielded similar editing efficacy throughout the brain. This may represent a candidate treatment to reduce the risk of AD.

The recent evolution of oncolytic virotherapy has yielded viral platforms with enhanced tumor tropism and expanded engineering flexibility, thereby enabling not only direct oncolysis but also deliberate promotion of antitumor immune responses. Here, we systematically explore multiple insertion sites for calreticulin (CALR) within an oncolytic adenovirus, identifying the most optimal variant that exposes robust CALR on tumor cell membrane and functionally motivates macrophages in addition to directly mediating tumor cell lysis. Mechanistically, this variant incorporates a precise deletion within the E1A CR2 domain (920–946 bp), enabling selective replication in retinoblastoma-deficient tumor cells. Furthermore, the E3-gp19k region has been replaced with an exogenous Cytomegalovirus (CMV) promoter to achieve precise regulation of CALR overexpression and diminish systemic toxicity. Functionally, by bulk-RNA-Seq, we demonstrate that oncolytic adenovirus (oAd)-CMV-CALR transfection induces endoplasmic reticulum stress, as evidenced by upregulation of phosphorylated eIF2α, which facilitates the translocation of CALR to the plasma membrane. Via ex vivo coculture assays, we validate that oAd-CMV-CALR transfection enhances the phagocytic capacity of M2 macrophages and promotes their repolarization toward an M1-like phenotype. These findings are further validated in patient-derived ovarian cancer spheroids, underscoring the translational potential of our approach. In vivo, oAd-CMV-CALR suppresses Hepa1-6 xenograft growth, boosts CD8⁺ T-cell infiltration, and exhibits favorable safety. Collectively, our findings highlight oAd-CMV-CALR as a potential therapeutic approach to modulate the tumor microenvironment and improve cancer immunotherapy outcomes.

Chimeric antigen receptor T (CAR-T) cells, created by gene editing systems along with recombinant adeno-associated virus (rAAV), provide a promising strategy for treating leukemia. rAAVs serve as a safe and effective donor template for homology-directed repair because they can avoid integrating into the host genome. However, only a few AAV serotypes can efficiently transduce human primary T cells at low multiplicities of infection (MOIs) with high packaging efficiency. To address this problem, variants derived from an AAV2 peptide library were screened in Jurkat cells and later validated in primary T cells. A high-ranking sequence identified outside the VR-VIII region, NNSKLTV, was discovered after three rounds of selection and was named Tot3. Tot3 demonstrated transduction efficiency similar to AAV2, but at a 27-fold lower MOI. In addition, Tot3 exhibited greater packaging efficiency and reduced thermal stability. Simultaneously, programmed cell death protein 1 (PD-1) knockout and CAR overexpression were achieved in human primary T cells using Tot3, with knockout and knock-in efficiencies reaching up to 70% and 55%, respectively. These CAR-T cells demonstrated significantly enhanced antitumor activity and increased survival times in a mouse model of diffuse B cell lymphoma.

Mutations in the Eyes Shut Homolog (EYS) gene are one of the leading causes of autosomal recessive retinitis pigmentosa, a progressive retinal degenerative disease for which no effective treatment currently exists. However, the large size of the EYS coding sequence (∼9.4 kb) exceeds the packaging limit of adeno-associated virus (AAV) vectors, posing a major barrier to gene replacement therapy. To address this challenge, we developed a tripartite AAV vector system that enables delivery and reconstitution of the full-length EYS gene using a Cre-lox-based unidirectional DNA recombination strategy, Uni-directional and Site-specific Transgene Assembly by Recombination (Uni-STAR). The system consists of three AAV constructs carrying discrete EYS segments flanked by engineered, noncompatible lox sites that drive ordered and unidirectional recombination in target cells. We validated this system in vitro by demonstrating successful reconstitution and expression of full-length EYS protein in HEK293T cells. In vivo, subretinal co-injection of the three AAV vectors into mouse eyes led to precise reconstitution and expression of full-length EYS protein in the retina. These findings establish the feasibility of using a tripartite AAV system to deliver the complete EYS gene and provide a foundation for future therapeutic development targeting EYS-associated retinal degenerations.