Differentiation and Advantages (AskBio's Toolbox)

While other AAV gene therapy companies have in-licensed a serotype for use for a particular therapeutic indication, or have a few capsid types available for use, AskBio has a wide array of AAV technology tools available. More importantly, if an ideal capsid does not exist, AskBio has the ability to create new capsids with specific performance attributes. This enables AskBio to target particular tissues, de-target other tissues, and reduce the limitations of neutralizing antibodies. As such, AskBio is not limited in the scope of what types of diseases it can treat and can minimize some of the detrimental attributes associated with other AAV capsids. Due to the expansiveness of its AAV capabilities, AskBio has been able to adopt an adaptive model for therapeutic development. AskBio learns from human clinical trial experiences and adapts AAV technology based on those learnings for enhanced performance.

AskBio’s unique and adaptive platform allows for continuous learning, improvement, and adaptation through best-in-class technology.

Hybrids

AAV is a non-enveloped virus containing a single-stranded DNA genome flanked by two complex DNA structures called ‘inverted terminal repeats’ (ITRs). These two terminal DNA structures originally obtained from the AAV2 serotype genome, are necessary to package the AAV recombinant genome (e.g., carrying the therapeutic genome) not only in an AAV2 capsid but also and strikingly into any serotype-specific vector capsid (e.g., type 1-12, chimeric, etc.). This ability to cross-package a recombinant AAV genome carrying the therapeutic expression cassette flanked by the ITRs from AAV2 serotype into any vector capsid, provides a highly flexible and rapid approach that simplifies research and development.

Self-Complementary Vectors

AAV is a non-enveloped virus containing a single-stranded DNA genome. Once the wild type or recombinant genome reaches the nucleus, it needs to be converted to a double-stranded DNA molecule capable of supporting transcription. This conversion step has been identified as being a major rate-limiting step in AAV-mediated gene expression. The development of novel engineered inverted terminal repeat sequences that generate self-complementary genomes (double-stranded) that are both packaged into capsids and also capable of supporting immediate transcription upon release in the nucleus has overcome this significant hurdle (McCarty DM, 2001). The result is that these novel “double-stranded” AAV vectors (also called ‘self-complementary’) may deliver a functional gene in cells that would not normally convert a single-stranded AAV genome to double-stranded DNA, as well as lead to faster onset and higher levels of gene expression. Also, these novel self-complementary AAV vectors counter balance abortive intranuclear pathways increasing the therapeutic index of each AAV particle. The ability to mediate robust and faster onset of vector transgene expression (e.g., hours versus days) allows for faster functional readout. A ten-to-one hundred-fold increase in efficiency of gene transfer from self-complementary AAV vectors over conventional ones was repeatedly observed in a large range of target tissues (McCarty et al., 2001 & 2003; Fu et al., 2003; Rehman et al., 2005; Xu et al., 2005; Nathwani et al., 2006 & 2007). The overall consequence in using self-complementary AAV vectors is that gene transfer will occur in a larger proportion of cells in a target tissue at a lower given dose than standard or naturally occurring single-stranded vectors. In summary, this technology provides for significantly increased gene transfer, earlier onset of gene expression, reduced manufacturing requirements, reduced safety concern and is compatible with all AAV serotypes evaluated to date.

Highly Efficient Duplexed AAV Genomes for Rapid-Onset Gene Expression
Adeno-associated virus (AAV) is a nonpathogenic, helper-dependent member of the parvovirus family. One of the identifying characteristics of this group is the encapsidation of a single-stranded DNA (ssDNA) genome. At each end of the ssDNA genome, a palindromic terminal repeat (TR) structure base-pairs upon itself into a hairpin configuration. This serves as a primer for cellular DNA polymerase to synthesize the complementary strand after uncoating in the host cell. Recombinant AAV (rAAV) gene delivery vectors also package ssDNA of plus or minus polarity and must rely on cellular replication factors for synthesis of the complementary strand. While it was initially expected that this step would be carried out spontaneously, by cellular DNA replication or repair pathways, this does not appear to be the case when testing in vivo (McCarty et al., 2003). Early work with rAAV vectors revealed that the ability to detect marker gene expression was dramatically enhanced when cells were co-infected with adenovirus, or transiently pretreated with genotoxic agents. This enhancement correlated with the formation of duplex DNA from the single-stranded virion DNA (vDNA) (Ferrari et al., 1996).

The requirement for complementary-strand synthesis, or recruitment, is now considered to be a limiting factor in the efficiency of rAAV vectors. Rather than rely on potentially variable cellular mechanisms to provide a complementary-strand for rAAV vectors, we have found that we can circumvent this rate-limiting problem by packaging both strands as a single (self-complementary) DNA molecule. We observed a ten-to-one hundred-fold increases in efficiency of transduction from scAAV vectors over conventional rAAV in a large range of target tissues (McCarty et,.al., 2001 & 2003, Fu et. al., Mol. Therapy 2003, Rehman et. al., Gene Therapy 2005, Xu et. al., Mol Therapy 2005, Nathwani et. al., Blood 2006, 2007). More importantly, unlike conventional single-stranded AAV vectors, inhibitors of DNA replication did not affect transduction from scAAV vectors (McCarty et al., Gene Therapy 2001). In addition, scAAV vectors display a rapid onset and a higher level of transgene expression in every tissue tested to date and have shown efficient transduction in vivo in tissue not transduced by conventional single-stranded AAV vector (Borras et. al., J. Gene Medicine 2006). These observations have now been reproduced in numerous animal models, including non-human primates (Nathwani et. al., Blood 2006, 2007). Thus, there is a shorter lead-time to the gene expression levels required for functional genomics and translational studies. Finally, the lower doses necessary to mediate a given level of gene expression offer manufacturing (reduced cost and production times) as well as pre-clinical & clinical benefits (lower doses will have improved safety profiles). All of these biological attributes have led to these duplexed vectors being treated as a new generation of AAV vectors that should significantly contribute to the ongoing development of parvovirus-based gene delivery systems.

In summary, this technology provides for significantly increased transduction efficiency, earlier onset of gene expression, reduced manufacturing requirements, and is compatible with all AAV serotypes evaluated to date.

Chimeric Capsids

Vectors based upon naturally occurring AAV serotypes have broad tropism, and therefore a potential of naturally delivering DNA to off-target tissues. These characteristics are the direct result of evolutionary pressures associated with natural route of infection and with the dependent association with a helper virus. Genetic engineering has allowed for the development of a new generation of AAV vectors – called chimeric – that have novel surface properties that are custom designed to fit a given therapeutic application. We have developed novel chimeric AAV capsids that mediate efficient gene delivery to a given tissue target while being de-targeted from tissues that normally accumulate the highest levels of AAV, while also possessing novel immunological properties.

We use the natural occurring serotypes as the starting materials to engineer chimeric vectors with specified performances characteristics. This technology provides the ability to design and construct a nearly infinite number of novel vector capsids with properties such as tissue/cell selectivity, immune system evasion, and characteristics including and surpassing those that may be obtained by use of the limited naturally occurring serotypes.

Our proprietary chimeric technique of designing capsid proteins derived from multiple AAV serotypes, and including capsid sequences from other viruses, has been shown to alter the natural tropism range of the resulting chimeric AAV vectors. These engineered vectors allow for targeting specific tissues/cells over others after systemic administration (e.g., Central Nervous System, Cardiac, Muscle, etc.), as well as the ability to de-target organs (e.g., de-targeting liver).

The chimeric technology also supports the development of products that may circumvent population limitations imposed by seroprevalence of neutralizing antibodies to naturally occurring serotypes. The first synthetic AAV vector (chimeric AAV2.5) developed for muscle-specific tropism with a unique immune profile was used in Phase-I studies for therapeutic gene delivery in Duchenne Muscular Dystrophy patients (Bowles DE, 2012).

Heparin and heparan sulfate binding chimeric vectors
This family of chimeric capsids contains modified receptor-recognition sites that change the cell or tissue tropism of the capsids. Modified heparin sulfate binding properties have been shown to mediate modified transduction of target cells in lung tissue.

Inner Loop chimeric vectors
This family of chimeric capsids with modified inner-loop regions have shown novel biodistribution and transduction patterns after systemic administration. Most if not all AAV vectors preferentially transduce the liver after systemic administration resulting in poor therapeutic index for diseases where the primary tissue targets are for instance heart, skeletal muscles, brain…Some inner-loop variants have the feature of significant 10-100x reduction of liver transduction while retaining high-level tropism for other target tissues (Asokan A., 2010; Shen S. 2013). These properties have encouraged the use of inner-loop mutants in clinical programs targeting cardiac muscle.

Dual Glycan receptor chimeric vectors
New AAV capsids can be evolved to recognize different host glycans through mutagenesis and experimental adaptation. These AAV capsid mutations affect viral binding to cells with carbohydrate receptors. So, we described the rational design and synthesis of a novel class of adeno-associated viruses (AAV) that exploit two distinct glycan receptors for cell entry. A prototypical dual glycan binding AAV strain was engineered by “grafting” a galactose (Gal) binding footprint from AAV serotype 9 onto the heparan sulfate binding AAV serotype 2. The resulting chimera, AAV2G9, interchangeably exploits Gal and HS as evidenced by competitive inhibition assays with lectins and glycans (Shen S., 2013). Further, AAV2G9 mediates rapid onset and sustained, higher transgene expression in vivo compared to parental AAV serotypes. In addition to demonstrating the modularity of glycan receptor footprints on viruses, our approach provides design parameters to upgrade the current AAV vector toolkit for clinical gene therapy.

Broad Patents and Intellectual Property Portfolio

The intellectual property landscape for AAV is crowded and confusing. Dozens of patents exist for nearly every step of AAV production, various vector capsids, and patents for clinical uses of the virus are appearing rapidly as well. For companies wishing to practice AAV-mediated gene therapy, royalty stacking can present a formidable challenge to profitability. AskBio’s comprehensive patent portfolio offers technical and, equally important, strategic advantages. Due to the breadth of the company’s portfolio of patents, covering all aspects of gene delivery, and numbering over three hundred patents, AskBio has become an extremely attractive partner for pharmaceutical and biotechnology companies intending to commercialize AAV-based gene therapy products. Due to the level of innovation at the company, it will not experience a patent cliff, but has an evergreen position.