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36 | SEPTEMBER 1, 2016 | GENengnews.com | Genetic Engineering & Biotechnology News Terence R. Flotte, M.D. Twenty-seven years after Steven A. Rosenberg, M.D., Ph.D., at the National Cancer Institute Bethesda campus performed the first human gene transfer experiment, gene therapy is poised to take its place in the armamentarium of biologic therapies available to treat human diseases. Some examples: the EMA-approval in November 2012 of Glybera ® (Uniqure), an intramuscular recombinant ade- no-associated virus (rAAV) product for treatment of lipo- protein lipase deficiency; and the recently announced Phase II trial results showing efficacy of another rAAV product for a genetic cause of blindness (LCA2)—for which a Biologics Licensing Application is anticipated in the U.S. this year. These two rAAV products emerging onto the mar- ket represent only the first trickle of what promises to be a gusher of new gene therapy products moving into the world of clinical medicine in the coming years. Sev- eral other remarkable examples of clini- cal efficacy from gene therapy have been reported. These include results of rAAV trials of products for hemophilia A and B and spinal muscular atrophy as well as tri- als of lentivirus-modified chimeric antigen receptor T cells (CAR-T) cells for a variety of cancers, most notably the anti-CD19 CAR-T therapy for leukemia. Additional gammaretrovirus and len- tivirus-based approaches to treat diseases affecting hematopoietic stem cells, such as X-SCID, ADA-SCID, WAS, and MLD have shown impressive clinical efficacy. In addition, a myriad of gene therapy approaches are making their way through preclinical and clinical proof-of-concept studies. These include rAAV-based gene therapies for other single gene disorders affecting the retina, the CNS, skeletal mus- cle, and liver. Also, the vector-mediated delivery of specific antibodies as a poten- tial therapy or prophylaxis has emerged in the treatment of infectious diseases such as influenza and HIV. The most impressive aspect of gene therapy in 2016, however, is the broad array of new research activity fur- ther up the pipeline, in terms of both new platform tech- nologies for gene modification and new proof-of-concept studies on how those platform technologies can be de- ployed to fight specific human diseases. New Genetic Therapy Platforms Since the dawn of gene therapy, when the Nobel-re c- ognized work of Varmus and Bishop on how gammaretro- viruses mediate tumorigenesis led to the first recombinant viral vectors, the scientific discoveries underpinning gene therapy have come from unexpected quarters. A few recent examples include the discovery of RNA interference as a means of vertically transmitted gene regulation in C. elegans by Fire and Mello, the discovery of the CRISPR/Cas9 system as a host defense system in S. pyogenes, and ongoing discoveries about the co-evo- lution between mammals and their viruses extending the pioneering work of Varmus, Bishop, and their colleagues. The discovery of RNAi mentioned above represented a paradigm shift for gene therapy because it allowed, for the first time, for truly robust, genetically targeted down-regulation of gene expression, which is crucial for the treatment of dominant genetic disorders and other conditions where disease is caused by over-activity of a detrimental gene product rather than by deficiency of a salutary one. Previous approaches, such as anti-sense oligo-deoxy- nucleotides (AS-ODN) and ribozymes, could, in principle, down-regulate genes by targeting specific sequences, but the efficiency of such systems was not high enough to allow for reliable translation beyond the tissue culture setting. RNAi, in contrast, was the first mechanism that com- bined nucleotide sequence-based specificity with a robust enzymatic activity, in this case the RNA-in- duced silencing complex (RISC)-complex. That combination of sequence-based tar- geting with enzymatic power turns out to be the key combination for all the systems that "work" in the sense of being efficient enough to achieve clinical results, once the inherent limitations of delivery technology come into play. The first iteration of such therapies to achieve clinical efficacy is small-interfering RNA (si-RNA), a technology for double- stranded RNA oligonucleotides that har- nesses the RNAi pathway to down-regu- late offending genes. Gal-Nac conjugates of siRNA have been pioneered by Alnylam Pharmaceuticals as a means to deliver any desired siRNA sequence to hepatocytes (Figure 1). This has been used as a therapy for transthyretin (TTR)-mediated amyloi- dosis, complement-mediated diseases, and other genetic and acquired conditions in which gene knockdown within hepato- cytes can interrupt the pathogenesis of the disease. Another technique for harnessing the RNAi pathway that may be more suitable for long-term knockdown in autosomal dominant genetic disorders is long-term expression of synthetic miRNA. The dis- covery of miRNA in C. elegans by Ambros and Ruvkun actually preceded that of ex- ogenous RNA-mediated down-regulation by Fire and Mello. The discovery that both modalities uti- lize the same enzymatic pathway has led to Gene Therapy 2016: The Pipeline Is Swelling TRANSLATIONAL MEDICINE 35th Anniversary Feature Door Has Been Opened to a Range of Novel Biological Therapies for Human Diseases Figure 1. siRNA-GalNAc conjugates have allowed for efficient knockdown of genes in the human liver. Reprinted with permission from Nair, J.K., et al., Multivalent N-acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing. J Am Chem Soc, 2014. 136(49): p. 16958-61. ©2014 American Chemical Society; Alnylam Pharmaceuticals Figure 2. Genome Editing with CRISPR/Cas9 Sontheimer, E.J. and R. Barrangou, The Bacterial Origins of the CRISPR Genome-Editing Revolution. Hum Gene Ther, 2015. 26(7): p. 413-24. Terence R. Flotte, M.D. (Terry.Flotte@ umassmed.edu), is the Celia and Isaac Haidak Professor in Medical Education, dean of the School of Medicine, provost, executive deputy chancellor, faculty mem- ber at the Horae Gene Therapy Center, and professor of pediatrics the University of Massachusetts Medical School. He is also the editor-in-chief of Human Gene Therapy, published by Mary Ann Liebert.