After almost 30 years of promise tempered by setbacks, gene therapies are rapidly becoming a critical component of the therapeutic armamentarium for a variety of inherited and acquired human diseases. Gene therapies for inherited immune disorders, hemophilia, eye and neurodegenerative disorders, and lymphoid cancers recently progressed to approved drug status in the United States and Europe, or are anticipated to receive approval in the near future. In this Review, we discuss milestones in the development of gene therapies, focusing on direct in vivo administration of viral vectors and adoptive transfer of genetically engineered T cells or hematopoietic stem cells. We also discuss emerging genome editing technologies that should further advance the scope and efficacy of gene therapy approaches.

CCR5 is the major co-receptor used by HIV-1 and individuals homozygous for a 32bp deletion in CCR5 are profoundly resistant to HIV-1 infection. Using engineered zinc finger nucleases (ZFNs), we were able to disrupt the CCR5 gene in human hematopoietic stem/progenitor cells (HSC) at a mean frequency of 17% of total alleles in a population. This procedure produces both mono and bi-allelically disrupted cells. ZFN-treated HSC retained the ability to engraft NOD/SCID/IL2rγnull mice and gave rise to polyclonal multi-lineage progeny with the CCR5 gene permanently disrupted. Control mice receiving untreated HSC and challenged with CCR5-tropic HIV-1 displayed profound CD4+ T cell loss. In contrast, mice transplanted with ZFN-modified HSC underwent rapid selection for CCR5-negative cells, had significantly lower HIV-1 levels and preserved human cells throughout their tissues. The demonstration that a minority of CCR5-modified HSC can populate an infected animal with HIV-1-resistant, CCR5-negative progeny suggests the use of ZFN-modified autologous HSC as a clinical approach to treating HIV-1.

Genetic modification of clinical-grade T cells is undertaken to augment function, including redirecting specificity for desired antigen. We and others have introduced a chimeric antigen receptor (CAR) to enable T cells to recognize lineage-specific tumor antigen, such as CD19, and early-phase human trials are currently assessing safety and feasibility. However, a significant barrier to next-generation clinical studies is developing a suitable CAR expression vector capable of genetically modifying a broad population of T cells. Transduction of T cells is relatively efficient but it requires specialized manufacture of expensive clinical grade recombinant virus. Electrotransfer of naked DNA plasmid offers a cost-effective alternative approach, but the inefficiency of transgene integration mandates ex vivo selection under cytocidal concentrations of drug to enforce expression of selection genes to achieve clinically meaningful numbers of CAR +

Green fluorescent protein (GFP) is a widely used intracellu-EGFP did lead to rapid development of disease in immunolar reporter molecule to assess gene transfer and deficient Nu/Nu mice. Mice surviving BM185/EGFP leukeexpression. A potential use for GFP is as a co-expressed mia challenge developed high cytotoxic T lymphocyte marker, to select and enrich gene-modified cells by flow (CTL) responses against EGFP-expressing cells. Furthercytometry. Processed peptides derived from GFP and more, immune stimulation against BM185/EGFP cells presented by the major histocompatibility complex on the could also be induced by immunization with EGFP+ transcell surface could potentially induce T cell immune duced dendritic cells. The effects of the co-expression of responses against GFP+ cells. Thus, clinical application of EGFP and immunomodulators (CD80 plus GM-CSF) were GFP is premature, since in vivo studies on its immunoalso investigated as an irradiated leukemia vaccine. EGFP genicity are lacking. Therefore, we investigated immune co-expression by the vaccine did not interfere with the responses against EGFP (enhanced-GFP) in two transdevelopment of CTLs against the parental leukemia or with plantable murine models: the BALB/c (H-2 that the immune response against EGFP may interfere with BM185 and EL-4 cell lines modified to express high levels its applicability in gene insertion/replacement strategies but of EGFP showed drastic reduction of disease development could potentially be employed for leukemia cell vaccines. when transplanted into immunocompetent mice. BM185/