Overcoming matrix effects in AAV neutralization assays with a constant serum concentration approach.

Strategies to Modulate Immune Responses: A New Frontier for Gene Therapy

Adeno-Associated Virus Vectors in Clinical Trials

Cationic Lipid Formulations Alter the In Vivo Tropism of AAV2/9 Vector in Lung

Self-complementary AAV Vectors; Advances and Applications

Gene therapy for colon cancer by adeno-associated viral vector-mediated transfer of survivin Cys84Ala mutant

Reversal of Blindness in Animal Models of Leber Congenital Amaurosis Using Optimized AAV2-mediated Gene Transfer

Adeno-associated viruses (AAV) have attracted significant attention in the field of gene and cell therapy due to highly effective delivery of therapeutic genes into human cells. The ability to generate recombinant AAV vectors compromised of unique or substituted protein sequences has led to the development of capsid variants with improved therapeutic properties. Seeking novel AAV vectors capable of enhanced transduction for therapeutic applications, we have developed a series of unique capsid variants termed AAV X-Vivo (AAV-XV) derived from chimeras of AAV12 VP1/2 sequences and the VP3 sequence of AAV6. These AAV variants showed enhanced infection of human primary T cells, hematopoietic stem cells, and neuronal cell lines over wildtype parental viruses, and superiority over AAV6 for genomic integration of DNA sequences by AAV alone or in combination with CRISPR gene editing. AAV-XV variants demonstrate transduction efficiency equivalent to AAV6 at multiplicities of infection 2 logs lower, enabling T cell engineering at low AAV doses. The protein coding sequence of these novel AAV chimeras revealed disruptions within the assembly-activating protein (AAP) which likely accounted for observed lower virus yield. A series of genome alterations, reverting the AAP sequence back to wildtype AAV6, had a negative impact on the enhanced transduction seen with AAV-VX, indicating overlapping functions within this sequence for both viral assembly and effective T cell transduction. Our findings show these AAV-XV variants are highly efficient at cell transduction at low dose and demonstrates the importance of the AAP coding region in both viral particle assembly and cell infection.IMPORTANCE A major hurdle to the therapeutic potential of AAV in gene therapy lies in achieving clinically meaningful AAV doses, and secondarily, ability to manufacture commercially viable titers of AAV to support this. By virtue of neutralizing antibodies against AAV that impede patient repeat-dosing, the dose of AAV for in vivo gene delivery has been high, which has resulted in unfortunate recent safety concerns and deaths in patients given higher-dose AAV gene therapy. We have generated new AAV variants possessing unique combinations of capsid proteins for gene and cell therapy applications termed AAV-XV, which have high levels of cell transduction and gene delivery at lower MOI. Furthermore, we demonstrate a novel finding, and an important consideration for recombinant AAV design, that a region of the AAV genome encoding the capsid viral protein and AAP is critical for both virus yield and the enhancement of infection/transduction.

Adeno-Associated Virus: A Ubiquitous Commensal of Mammals

Gene therapy using adeno-associated virus (AAV) vectors is currently expanding to broad clinical applications. As the presence of a neutralizing antibody (NAb) against AAV capsids significantly restrains their efficacy, an accurate evaluation of NAb status is crucial for selecting appropriate candidates for gene therapy. Notably, cell-based NAb assays may not be sufficiently sensitive for detecting low-titer NAb, and few assays can evaluate multiple AAV serotypes using a commonly available cell. In this study, we developed a sensitive NAb assay against various AAV serotypes using commonly available HEK293 and Huh-7 cells. We found that adding glucose efficiently enhanced transgene expression across various AAV serotypes without causing cell damage. In addition, by combining a highly sensitive reporter gene, NanoLuc, the necessary dose of AAV vector was significantly reduced. The reduction of AAV dose resulted in the increased sensitivity of NAb detection as low as 100 vector genomes/cell. At the lower vector doses, sensitivity improvement was not observed regardless of serotypes, suggesting the limit of assay sensitivity of the cell-based NAb assay. These findings provide a highly sensitive methodology for assessing NAb titers and offer insights into conditions to attain maximal sensitivity in the cell-based NAb assay.

B cell focused transient immune suppression protocol for efficient AAV readministration to the liver

Adeno-associated Virus-mediated, Mifepristone-regulated Transgene Expression in the Brain

Adeno-associated viruses (AAVs) exhibit a complex biphasic life cycle in cell culture, with productive replication dependent upon co-infection with a helper virus, whereas in the absence of a helper virus AAV latency is established. While vectors based on adeno-associated viruses are already widely used in human gene therapy, open questions remain concerning the regulation of AAV productive and latent infection, especially in the in vivo situation, for which no suitable animal model exists so far. To fill this gap, a combination of in vitro and in silico analyses were chosen in the present thesis to perform a comprehensive analysis of the AAV transcriptome based on state of the art, high-throughput total genomic RNA sequencing. The analysis of the transcription profiles and kinetics of the AAV (+) strand largely confirmed a variety of previous studies performed with classical RNA methods. However, several novel AAV splice variants were identified, the most abundant of which led to the expression of an 18 kDa Rep/VP fusion protein. Genetic analyses with corresponding virus mutants unable to express the newly identified splice variants showed that these have a major impact on early AAV replication steps, highly suggestive of an important role for in vivo AAV propagation. Maybe even more interesting, the AAV transcriptome analysis revealed for the first time the expression of transcripts derived from the AAV (-) strand. These transcripts were encoded in the p5 promoter region, which drives the expression of the large AAV2 regulatory proteins Rep78/68. The pattern of p5 sense and antisense transcription was highly reminiscent of a phenomena described in recent years as bidirectional transcription. This adds a further possible mechanistic level to the regulation of p5 driven gene expression, which represents a major switch point between productive and latent AAV infection. To identify small RNAs with regulatory roles in the AAV life cycle, the first AAV small RNA-Seq analysis was performed in AAV infected cells. A variety of novel AAV2-specific small RNAs located close to or within the AAV- ITRs was identified, which are apparently transcribed from the newly defined anti-p5 promoter. Validation on Northern blots and by argonaute Co-IP indicated alternative small RNA processing, divergent from the canonical miRNA pathway. In addition, small AAV2-specific RNAs in close proximity to viral promoters were suggestive of small promoter-associated RNAs that might act as transcriptional regulators during AAV latent and productive infection. Furthermore, the cellular miRNA expression profile showed no alteration upon AAV infection reassuring the long-established apathogenic nature of wild-type AAV. As a further prerequisite for a better understanding of AAV vector biology, the individual HSV helper genes for productive AAV5 replication were analyzed in a comparative study of the AAV2 and AAV5 replication cycle. This analysis revealed that the same combination of HSV1 helper genes identified for AAV2, promote AAV5 productive replication.

Biochemical, Pathological, and Skeletal Improvement of Mucopolysaccharidosis VI After Gene Transfer to Liver but Not to Muscle

In the past HIV infection was a common complication of haemophilia therapy. Gene therapy trials in Haemophilia patients using rAAV have shown promising results; Unfortunately, the majority of gene therapy trials studies have excluded HIV positive patients. We decided to systematically review the published clinical trials using rAAV for HIV prevention. A comprehensive literature search was performed to identify studies evaluating clinical trials using rAAV for HIV. The search was conducted using the MEDLINE/PubMed databases. Search keywords included 'gene therapy', 'adeno-associated virus', 'HIV' and 'clinical trial'. Three studies met our inclusion criteria. Two were phase 1 studies and one was a phase 2 study. One study examined an AAV coding for human monoclonal IgG1 antibody whereas the other two studies delivered a vector coding for viral protease and part of reverse transcriptase. All studies administered the vaccine intramuscularly and showed a response as well a good safety profile. The concept of using a viral vector to prevent a viral infection is revolutionary. Due to the paucity of information regarding application of any gene therapy in HIV patients and the potential use of gene therapy in haemophilia patients with HIV in the future warrants attention.

Long-Term Real-World Safety and Efficacy of Liver-Directed AAV Gene Therapy for Severe Hemophilia with and without Inbibitors in Privately Owned Dogs