Throughout its 40-year history, the field of gene therapy has been marked by many transitions. It has seen great strides in combating human disease, has given hope to patients and families with limited treatment options, but has also been subject to many setbacks. Treatment of patients with this class of investigational drugs has resulted in severe adverse effects and, even in rare cases, death. At the heart of this dichotomous field are the viral-based vectors, the delivery vehicles that have allowed researchers and clinicians to develop powerful drug platforms, and have radically changed the face of medicine. Within the past 5 years, the gene therapy field has seen a wave of drugs based on viral vectors that have gained regulatory approval that come in a variety of designs and purposes. These modalities range from vector-based cancer therapies, to treating monogenic diseases with life-altering outcomes. At present, the three key vector strategies are based on adenoviruses, adeno-associated viruses, and lentiviruses. They have led the way in preclinical and clinical successes in the past two decades. However, despite these successes, many challenges still limit these approaches from attaining their full potential. To review the viral vector-based gene therapy landscape, we focus on these three highly regarded vector platforms and describe mechanisms of action and their roles in treating human disease.

Incorporating TLR9-inhibitory sequences in the AAV vector genome inhibits immunogenicity and enhances transgene expression in multiple animal models.

The field of gene therapy has made considerable progress over the past several years. Adeno-associated virus (AAV) vectors have emerged as promising and attractive tools for in vivo gene therapy. Despite the recent clinical successes achieved with recombinant AAVs (rAAVs) for therapeutics, host immune responses against the vector and transgene product have been observed in numerous preclinical and clinical studies. These outcomes have hampered the advancement of AAV gene therapies, preventing them from becoming fully viable and safe medicines. The human immune system is multidimensional and complex. Both the innate and adaptive arms of the immune system seem to play a concerted role in the response against rAAVs. While most efforts have been focused on the role of adaptive immunity and developing ways to overcome it, the innate immune system has also been found to have a critical function. Innate immunity not only mediates the initial response to the vector, but also primes the adaptive immune system to launch a more deleterious attack against the foreign vector. This Review highlights what is known about innate immune responses against rAAVs and discusses potential strategies to circumvent these pathways.

Background: Osteogenesis is a highly regulated developmental process and continues during the turnover and repair of mature bone. Runx2, the master regulator of osteoblastogenesis, directs a transcriptional program essential for bone formation through genetic and epigenetic mechanisms. While individual Runx2 gene targets have been identified, further insights into the broad spectrum of Runx2 functions required for osteogenesis are needed. Results: By performing genome-wide characterization of Runx2 binding at the three major stages of osteoblast differentiation - proliferation, matrix deposition and mineralization - we identify Runx2-dependent regulatory networks driving bone formation. Using chromatin immunoprecipitation followed by high-throughput sequencing over the course of these stages, we identify approximately 80,000 significantly enriched regions of Runx2 binding throughout the mouse genome. These binding events exhibit distinct patterns during osteogenesis, and are associated with proximal promoters and also non-promoter regions: upstream, introns, exons, transcription termination site regions, and intergenic regions. These peaks were partitioned into clusters that are associated with genes in complex biological processes that support bone formation. Using Affymetrix expression profiling of differentiating osteoblasts depleted of Runx2, we identify novel Runx2 targets including Ezh2, a critical epigenetic regulator; Crabp2, a retinoic acid signaling component; Adamts4 and Tnfrsf19, two remodelers of the extracellular matrix. We demonstrate by luciferase assays that these novel biological targets are regulated by Runx2 occupancy at non-promoter regions. Conclusions: Our data establish that Runx2 interactions with chromatin across the genome reveal novel genes, pathways and transcriptional mechanisms that contribute to the regulation of osteoblastogenesis.

RNAi-based bone anabolic gene therapy has demonstrated initial success, but many practical challenges are still unmet. Here, we demonstrate that a recombinant adeno-associated virus 9 (rAAV9) is highly effective for transducing osteoblast lineage cells in the bone. The adaptor protein Schnurri-3 (SHN3)is a promising therapeutic target for osteoporosis, as deletion ofshn3prevents bone loss in osteoporotic mice and short-term inhibition ofshn3in adult mice increases bone mass. Accordingly, systemic and direct joint administration of an rAAV9 vector carrying an artificial-microRNA that targetsshn3(rAAV9-amiR-shn3) in mice markedly enhanced bone formation via augmented osteoblast activity. Additionally, systemic delivery of rAAV9-amiR-shn3in osteoporotic mice counteracted bone loss and enhanced bone mechanical properties. Finally, we rationally designed a capsid that exhibits improved specificity to bone by grafting the bone-targeting peptide motif (AspSerSer)₆onto the AAV9-VP2 capsid protein. Collectively, our results identify a bone-targeting rAAV-mediated gene therapy for osteoporosis.