Although cord blood transplantation has significantly extended the lifespan of mucopolysaccharidosis type 1 (MPS1) patients, over 95% manifest cornea clouding with about 50% progressing to blindness. As corneal transplants are met with high rejection rates in MPS1 children, there remains no treatment to prevent blindness or restore vision in MPS1 children. Since MPS1 is caused by mutations in idua, which encodes alpha-L-iduronidase, a gene addition strategy to prevent, and potentially reverse, MPS1-associated corneal blindness was investigated. Initially, a codon optimized idua cDNA expression cassette (opt-IDUA) was validated for IDUA production and function following adeno-associated virus (AAV) vector transduction of MPS1 patient fibroblasts. Then, an AAV serotype evaluation in human cornea explants identified an AAV8 and 9 chimeric capsid (8G9) as most efficient for transduction. AAV8G9-opt-IDUA administered to human corneas via intrastromal injection demonstrated widespread transduction, which included cells that naturally produce IDUA, and resulted in a >10-fold supraphysiological increase in IDUA activity. No significant apoptosis related to AAV vectors or IDUA was observed under any conditions in both human corneas and MPS1 patient fibroblasts. The collective preclinical data demonstrate safe and efficient IDUA delivery to human corneas, which may prevent and potentially reverse MPS1-associated cornea blindness.

Adeno-associated virus (AAV) has been isolated from numerous vertebrate species since 1966. Besides its wide and promiscuous tropism, AAV infection does not result in considerable toxicity or pathogenicity and is capable of achieving adequate and long-term levels of gene transfer, especially following generation of the AAV recombinant variant: rAAV. Due to these properties, rAAV has gained special attention as a viral vector for gene therapy in the last decade. Currently, there are 130 clinical trials taking place worldwide for several diseases testing the safety and efficacy profiles of rAAV. During preclinical and clinical studies, several challenges have arisen in terms of reaching the full therapeutic potential of rAAV, such as efficient delivery of the virus in a targeted and specific manner to a desired tissue. Importantly, the development of immune responses towards the viral capsids poses an obstacle to rAAV applicability in the clinical setting. Numerous approaches have been developed in order to tailor an optimized therapeutic virus for treating specific diseases, including the use of different AAV serotypes or the creation of recombinant capsid variants with distinctive transduction and immunological profiles. This chapter reviews current information on rAAV clinical trials and the potential for combining rAAV platform with other technologies, such as induced pluripotent cells and gene editing.

Mesenchymal stromal cells (MSC) constitutively express low levels of human leukocyte antigen-G (HLA-G), which has been shown to contribute to their immunomodulatory and anti-inflammatory properties. Here, we hypothesized that overexpression of HLA-G on bone marrow-derived MSC would improve their immunomodulatory function, thus increasing their therapeutic potential. Therefore, we investigated which gene transfer system is best suited for delivering this molecule while maintaining its immuno-modulatory effects. We performed a side-by-side comparison between three nonviral plasmid-based platforms (pmax-HLA-G1; MC-HLA-G1; pEP-HLA-G1) and a viral system (Lv-HLA-G1) using gene transfer parameters that yielded similar levels of HLA-G1-expressing MSC. Natural killer (NK) cell-mediated lysis assays and T cell proliferation assays showed that MSC modified with the HLA-G1 expressing viral vector had significantly lower susceptibility to NK-lysis and significantly reduced T cell proliferation when compared to nonmodified cells or MSC modified with plasmid. We also show that, in plasmid-modified MSC, an increase in Toll-like receptor (TLR)9 expression is the mechanism responsible for the abrogation of HLA-G1’s immunomodulatory effect. Although MSC can be efficiently modified to overexpress HLA-G1 using viral and nonviral strategies, only viral-based delivery of HLA-G1 is suitable for improvement of MSC’s immunomodulatory properties.

Despite having the best safety profile of any current clinical viral vector, it is known that AAV preps contain contaminating sequences that are packaged alongside the expression cassette at a low rate. These sequences can originate from production plasmid DNA, or chromosomal DNA from producer cell lines. It has been reported