Increasing the Specificity of AAV-Based Gene Editing through Self-Targeting and Short-Promoter Strategies.

Our group previously used adeno-associated viral vectors (AAVs) to express an engineered meganuclease specific for a sequence in the PCSK9 gene (M2PCSK9), a clinical target for treating coronary heart disease. Upon testing this nuclease in non-human primates, we observed specific editing characterized by several insertions and deletions (indels) in the target sequence as well as indels in similar genomic sequences. We hypothesized that high nuclease expression increases off-target editing. Here, we reduced nuclease expression using two strategies. The first was a self-targeting strategy that involved inserting the M2PCSK9 target sequence into the AAV genome that expresses the nuclease and/or fusing the nuclease to a specific peptide to promote its degradation. The second strategy used a shortened version of the parental promoter to reduce nuclease expression. Mice administered with these second-generation AAV vectors showed reduced PCSK9 expression due to the nuclease on-target activity and reduced off-target activity. All vectors induced a stable reduction of PCSK9 in primates treated with self-targeting and short-promoter AAVs. Compared to the meganuclease-expressing parental AAV vector, we observed a significant reduction in off-target activity. In conclusion, we increased the in vivo nuclease specificity using a clinically relevant strategy that can be applied to other genome-editing nucleases.

CRISPR/Cas9-mediated in vivo gene targeting corrects hemostasis in newborn and adult factor IX-knockout mice.

Many genetic diseases, including hemophilia, require long-term therapeutic effects. Despite the initial success of liver-directed adeno-associated virus (AAV) gene therapy for hemophilia in clinical trials, long-term sustained therapeutic effects have yet to be seen. One explanation for the gradual decline of efficacy over time is that the nonintegrating AAV vector genome could be lost during cell division during hepatocyte turnover, albeit at a slow pace in adults. Readministering the same vector is challenging as a result of the AAV-neutralizing antibodies elicited by the initial treatment. Here, we investigated the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated homology-directed gene targeting for sustained treatment of hemophilia B. We developed a donor vector containing a promoterless partial human factor IX (FIX) complementary DNA carrying the hyperactive FIX Padua mutation. A single injection of dual AAV vectors in newborn and adult FIX-knockout (FIX-KO) mice led to stable expression of FIX at or above the normal levels for 8 months. Eight weeks after the vector treatment, we subjected a subgroup of newborn and adult treated FIX-KO mice to a two-thirds partial hepatectomy; all of these animals survived the procedure without any complications or interventions. FIX levels persisted at similar levels for 24 weeks after partial hepatectomy, indicating stable genomic targeting. Our results lend support for the use of a CRISPR/Cas9 approach to achieve lifelong expression of therapeutic proteins.

Prevalent and Disseminated Recombinant and Wild-Type Adeno-Associated Virus Integration in Macaques and Humans.

Integration of naturally occurring adeno-associated viruses (AAV; wild-type AAV [wtAAV]) and those used in gene therapy (recombinant AAV [rAAV]) into host genomic DNA has been documented for over two decades. Results from mouse and dog studies have raised concerns of insertional mutagenesis and clonal expansion following AAV exposure, particularly in the context of gene therapy. This study aimed to characterize the genomic location, abundance, and expansion of wtAAV and rAAV integrations in macaque and human genomes. Using an unbiased, next-generation sequencing-based approach, we identified the genome-wide integration loci in tissue samples (primarily liver) in 168 nonhuman primates (NHPs) and 85 humans naïve to rAAV exposure and 86 NHPs treated with rAAV in preclinical studies. Our results suggest that rAAV and wtAAV integrations exhibit similar, broad distribution patterns across species, with a higher frequency in genomic regions highly vulnerable to DNA damage or close to highly transcribed genes. rAAV exhibited a higher abundance of unique integration loci, whereas wtAAV integration loci were associated with greater clonal expansion. This expansive and detailed characterization of AAV integration in NHPs and humans provides key translational insights, with important implications for the safety of rAAV as a gene therapy vector.

A mutation-independent CRISPR-Cas9-mediated gene targeting approach to treat a murine model of ornithine transcarbamylase deficiency.

Ornithine transcarbamylase (OTC) deficiency is an X-linked urea cycle disorder associated with high mortality. Although a promising treatment for late-onset OTC deficiency, adeno-associated virus (AAV) neonatal gene therapy would only provide short-term therapeutic effects as the non-integrated genome gets lost during hepatocyte proliferation. CRISPR-Cas9-mediated homology-directed repair can correct a G-to-A mutation in 10% of OTC alleles in the livers of newborn OTC spfash mice. However, an editing vector able to correct one mutation would not be applicable for patients carrying different OTC mutations, plus expression would not be fast enough to treat a hyperammonemia crisis. Here, we describe a dual-AAV vector system that accomplishes rapid short-term expression from a non-integrated minigene and long-term expression from the site-specific integration of this minigene without any selective growth advantage for OTC-positive cells in newborns. This CRISPR-Cas9 gene-targeting approach may be applicable to all patients with OTC deficiency, irrespective of mutation and/or clinical state.

Accurate and Rapid Sequence Analysis of Adeno-Associated Virus Plasmids by Illumina Next-Generation Sequencing.

Sequence validation of plasmid DNA is a crucial quality control step that must occur prior to adeno-associated virus (AAV) vector packaging through plasmid transfection. AAV cis-plasmids present unique challenges to sequence analysis, as they contain inverted terminal repeats and are prone to sequence rearrangements. An accurate and rapid next-generation sequencing approach has been established to analyze full-length sequences of AAV cis-plasmids within 3.5 days. Here, a step-by-step protocol is described that can reliably detect and identify the location and frequency of sequence variants commonly observed in AAV cis-plasmids.