Lentiviral vectors have revolutionized gene therapy by efficient and stable transduction of dividing and non-dividing cells, their large packaging capacity, and their compatibility with pseudotyping to alter viral tropism. The vesicular stomatitis virus glycoprotein (VSV-G) is widely used as a viral envelope protein of choice to pseudotype lentiviral vector particles as it confers exceptional particle stability and a broad tropism, due to the ubiquitous nature of the low-density lipoprotein receptor (LDLR). While this broad tropism facilitates transduction of diverse cell types, it precludes accurate in vivo targeting of specific cell populations. Structural insights into VSV-G have made receptor-blinding possible and revealed sites amenable to mutation while preserving fusion capacity. Coupled with targeting moieties, VSV-G pseudotyped lentiviral particles are redirected towards cells expressing target antigens. Such targeted vectors open new possibilities for in vivo gene therapy across oncology, infectious diseases, transplantation medicine, and other diseases. Use of targeted vectors will make in vivo gene therapy more accessible than cost-intensive ex vivo gene therapies. Since targeted vectors will be available as 'off-the-shelf' drugs, they will also drastically reduce time-to-treatment. This review highlights advances in bioengineering to exploit the versatility of VSV-G-pseudotyped lentiviral vectors and explores their vast potential for targeted gene delivery.
This review provides a comprehensive overview of the current status of viral and non-viral vector systems for in vivo and ex vivo applications, and key comparisons are made across safety, efficacy, scalability, and immune responses.
Chimeric antigen receptor T cell immunotherapies are transforming therapies for hematological malignancies and solid tumors and can be enhanced by targeted gene knockout. Here, we report lentiviral-based virus-like particles that package and deliver Cas9 ribonucleoproteins to primary human T cells. Using distinct pseudotyping strategies for virus-like particles and for lentiviral or γ-retroviral vectors, we achieved chimeric antigen receptor expression and targeted gene disruption. Under optimized transduction conditions, more than 50% of T cells expressed a chimeric antigen receptor by flow cytometry, with vector copy numbers exceeding two. Editing efficiencies were above 70% at three different target loci tested: T cell receptor α constant chain, β2-microglobulin, and DNA methyltransferase 3α. When the editing efficiency of virus-like particles was directly compared to electroporation, electroporation achieved a higher editing efficiency (99% versus 70%–90%). However, virus-like particle treatment resulted in twice as many cells being recovered compared with electroporation with a 10% increase in cell viability. Furthermore, off-target editing in virus-like particle-treated cells was reduced compared to ribonucleoprotein electroporated cells. These results support the feasibility of using virus-like particle-mediated delivery of Cas9 ribonucleoprotein to disrupt genes of interest, enabling a more scalable and cost-effective process for generating T cell immunotherapies.
Francesca Ferrara, Matthew M Wielgosz, Jeoungeun J. Park et al.· Molecular therapy. Advances· 0 citations
Targeted in vivo transduction, entailing direct administration of viral vector preparations to patients, is the next big step in gene therapy. To redirect lentiviral vector (LV) particles selectively to desired cell type(s), different glycoproteins have been engineered to alter their tropism, including the glycoprotein G from Vesicular Stomatitis Virus (VSV-G) as the most commonly employed tropism-defining protein for LV particles. For detargeting from its natural receptor, the low-density lipoprotein receptor (LDLR), a VSV-G variant with two blinding substitutions, K47Q and R354A, is commonly used (VSV-G.pub). We provide first evidence for insufficient blinding of VSV-G.pub in human and murine cell lines and primary cells. In silico modeling pointed toward only slightly reduced LDLR binding affinity of VSV-G.pub. To lower the affinity further, we generated three novel VSV-G variants based on charge-reversing substitutions in two, four, or six key residues. These variants achieved a more stringent blinding compared with VSV-G.pub in the tested cell lines and primary cells. Codisplay of a CD4 binder enabled lentiviral particles pseudotyped with the novel variants to selectively transduce CD4-expressing cells. In summary, we present improved VSV-G variants with a better on/off-target ratio as attractive tools for in vivo gene therapy applications.
Felix L. Warnecke, M. Ertelt, Anjali Shrivastava et al.· Human Gene Therapy· 0 citations
A virus-like particle (VLP)-based toolkit that delivers diverse CRISPR editing modalities to human monocytes, macrophages and dendritic cells with high efficiency while preserving viability and innate immune responsiveness is presented.
Hyuncheol Jung, Pascal Devant, Carter Ching et al.· Nature Biotechnology· 0 citations
Several viral vectors have been developed for gene therapy due to their high transduction efficiency, but some integrate into the host genome, raising safety concerns. Recent studies have identified recombinant adeno-associated virus serotype 6 (rAAV6) as a promising vector for hematopoietic stem and progenitor cell-targeted gene therapy because of its non-pathogenic nature, low integration frequency, and capacity for sustained episomal transgene expression. Nevertheless, its chromosomal integration profile remains incompletely defined, warranting a comprehensive evaluation to assess long-term safety. In this study, human CD34＋ cells were transduced with rAAV6 under varying vector doses and transgene contexts, and integration-site mapping was performed using the integration-site enriched library sequencing approach. Consistent with the largely episomal nature of AAV, high vector sequence alignment rates were observed across all groups. rAAV6 integrations occurred randomly throughout the genome, showing a broad pan-chromosomal distribution without evidence of sequence-specific targeting or clustering. Although integrations were more frequent in CpG islands, commonly located within open chromatin, this pattern likely reflects chromatin accessibility rather than targeting bias. Functional enrichment analysis indicated associations with general cellular and structural processes, without enrichment in oncogenic pathways. Distance-based analysis confirmed that integration sites were mapped at a distance from oncogenes and tumor suppressor genes, even under high-dose conditions. The data support the genomic safety of rAAV6 and its applicability to hematological gene therapy.
H. Lee, Nayoung Park, In-Byung Park et al.· International Journal of Ste...· 0 citations
Clinical successes in gene therapy using adeno-associated vectors (AAV) are offering a hopeful path toward the correction of several monogenic disorders. At its core, AAV gene delivery relies on multiple interactions between the capsid or vector genome and numerous host cell factors, a complex process that remains incompletely understood. Whether different serotypes similarly hijack the intracellular machinery in a cell, and whether these processes are necessarily conserved across species, are not known. To identify host factors enabling or preventing transduction across species or serotypes, CRISPR/Cas9 whole-genome knockdown screens were conducted in mouse (AML12) and human (HuH-7) cell lines before transduction with AAV2 or AAV9. Key common entry factors (AAVR, GPR108) were confirmed, with many other serotype- and/or species-specific host cell modulators identified. Interestingly, while serotype-specific differences were observed for successful transduction of a specific cell line, species-specific divergences were even more striking when comparing results across murine and human cells for the same serotype. Leveraging these data, we developed a novel approach to detarget liver transduction by transiently downregulating the expression of key entry factors in this tissue using GalNac-siRNAs prior to AAV9 administration. Such manipulation not only blunted hepatic transduction but also redirected the vector to other transduction-permissive tissues.
Katie Kubek-Luck, J. Velazquez, Xiaorui Yao et al.· Molecular Therapy· 0 citations