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Welcome to the Gene Therapy Program
Providing a foundation for basic research necessary to assure the success of gene therapy.
Given all the developments in molecular genetics, the isolation and cloning of genes is now a relatively common procedure. Research now centers on somatic gene therapy, referring to the techniques used to insert a functioning gene into the somatic (non-reproductive) cells of a patient to correct an inborn genetic error or to provide a new function to the cell. Having individual genes available opens the way for gene therapy to take place. And yet, after an initial period of about six years of preclinical work and another thirteen years involving clinical trials, effective gene delivery still remains one of the central challenges in the field.
The Gene Therapy Program of the University of Pennsylvania comprises basic scientific research and core lab research services. Our focus is on developing effective gene vectors derived from recombinant viruses. Much of our current effort is in the development of new adeno-associated virus (AAV) vectors, although some of our research involves both adenoviruses and lentiviruses. Several basic science core laboratories work together to support the development of new vectors.
AAV 3.0TM – From Bench to Bedside and Back
The laboratory of Jim Wilson at the Gene Therapy Program (GTP) of the University of Pennsylvania has been a leader in the development of innovative vector technology for close to three decades, having contributed to the development of a variety of non-viral and viral platforms. Their emphasis on in vivo gene therapy led them to focus on vectors based on adeno-associated viruses (AAVs). The current effort called AAV 3.0TM represents their most ambitious commitment to vector innovation so far. The goal is to create AAV vectors with substantially improved performance profiles for traditional gene therapy applications, as well as to identify AAV vectors suitable for genome editing.
AAVs were discovered in the 1960s as contaminants of laboratory preparations of adenoviruses. Six AAV serotypes emerged from this early work (Fig. 1). The first AAV vectors were created based on AAV serotype 2 (AAV2). These vectors were shown in mouse models to confer stable expression in non-dividing cells when injected in vivo without integrating into the chromosome. In addition, in vivo transduction was achieved without activating adaptive immune responses, providing a much-improved safety profile compared to other viral and non-viral vectors. A major limitation with AAV2 vectors, however, was that transduction efficiency was low for most cells with some exceptions, such as epithelial cells of the retina. Faculty at the University of Pennsylvania, Jean Bennett and Al Maguire, used AAV2 vectors to treat patients with a rare form of inherited blindness, which, depending on the outcome of current late stage clinical trials, could be the first approved gene therapy product in the US.
In the mid-1990s, the Wilson laboratory sought to improve the performance of AAVs by developing vectors based on other known serotypes, such as AAV1. They showed that AAV1 had improved transduction in skeletal and cardiac muscle. In fact, a novel therapeutic employing the AAV1 vector was developed for the treatment of an inherited form of high triglycerides by UniQure, which became the first gene therapy approved in Europe. Jay Chiorini from the National Institutes of Health (NIH) developed a vector based on AAV5, which also had improved transduction profiles. We refer to this early development of AAV vectors as AAV 1.0 (Fig. 1).
Because of disappointing clinical studies conducted in the late 1990s using existing viral and non-viral vectors, the Wilson laboratory and the University of Pennsylvania made a substantial commitment to the development of improved vectors. Their strategy was to leverage the favorable safety profiles of AAVs that emerged from studies using AAV 1.0 technology, but to make them more efficient in terms of in vivo gene therapy. In work that began in the 2000 timeframe, they identified AAVs from natural sources to capture a broader range of structural and functional diversity that occurs during natural in vivo infections.
Using molecular techniques they were able to identify a large and diverse family of endogenous AAVs which, when developed as vectors, were substantially improved compared to AAV 1.0. This new technology platform, which we designated AAV 2.0 (Fig. 1, also referred to as NAVTM) was widely and freely distributed to the academic community and licensed to REGENXBIO, a company founded by Penn to enable the commercialization of the platform. Subsequent sublicensing of the AAV 2.0 platform to 9 biotech partners has led to the development of 28 product candidates mostly targeting rare diseases with high unmet need, and has accounted for approximately 70 percent of all AAV gene therapy clinical trials relating to new treatment INDs posted on the U.S. government clinical trials database from 2012 through 2014.
A number of academic and commercial laboratories have attempted to improve the performance of AAV vectors since the creation of the AAV 2.0 platform. It is difficult to know whether these incremental efforts have yielded substantial improvements in efficiency and safety over the original AAV 2.0 vectors identified by the Wilson Lab and Penn since very little research has been done with these new vectors in large animals or humans. In the case of biopharmaceutical efforts, insufficient information is disclosed to allow for independent confirmation of relative performance.
Despite the tremendous progress that has been made in the last 25 years in AAV research, and the clinical successes that have and will likely continue to emerge from the use of AAV 1.0 and AAV 2.0 vectors, we believe there is a significant opportunity to further improve the performance of the AAV platform for gene therapy and to successfully apply it to a broader range of therapeutic applications using novel techniques such as genome editing. The Wilson laboratory and the University of Pennsylvania plan to make a substantial commitment toward achieving these goals through the development of AAV 3.0TM (Fig. 1).
What are the primary objectives of the AAV 3.0TM research?
We have two main goals. First, we will attempt to improve the performance of AAVs for standard applications of gene therapy focusing on enhanced delivery and potency, while circumventing pre-existing and induced humoral immunity without affecting the ease or scale of manufacturing. We will also develop AAVs to be more suitable for genome editing, which requires targeting of stem-like and progenitor cells rather than post-mitotic cells, which are the target of gene replacement therapy.
How will AAV 3.0TM differ from the many AAV development programs currently underway?
Current efforts in vector discovery utilize two general approaches: isolation of endogenous AAV from primates using techniques similar to that used by the Wilson lab in the discovery of AAV 2.0, and generation of a diversity of AAV capsid sequences in vitro, and selection of those with desired properties (primarily transduction efficiency) in vitro and more recently in vivo. Unfortunately, in these types of experiments, little is learned about why some vectors perform while others do not. While this work may lead to incremental improvements, it is not yielding the transformative advances that arose out of AAV 1.0 and AAV 2.0. With our newly announced AAV 3.0TM program, we will deploy modern techniques of molecular, structural and cell biology, and bioinformatics in three parallel, but interdependent, lines of research. Importantly, we will attempt to understand mechanisms of in vivo transduction to better inform technology development. The three lines of discovery research are:
- Isolation of AAVs from natural sources. We believe that the family of primate AAVs discovered under AAV 2.0 (and extended by others) only scratches the surface of AAV biology that exists in the wild. We will try to capture the full spectrum of endogenous AAVs in primate and non-primate species. Unique capsids will be evaluated in animal models, including primates.
- Molecular evolution of AAVs. Studies will be conducted to isolate improved AAVs using techniques of molecular evolution and in vivo selection. We do so, however, with appropriate caution. One of the most important features of the biology of AAV vectors derived from endogenous viruses is the lack of immune reactivity/toxicity in the host. Wild-type AAVs have created an ecological niche that allows their persistence and transmission with a unique biology characterized by low inflammation and active suppression of adaptive immune responses. This immune privilege drew us to AAVs in the first place and must be retained in next-generation vectors. Current selection methods for engineered capsids are driven by tropism and not immune activation. Our approach is to use existing information to focus mutagenesis in relevant hypervariable regions to create capsid libraries, which are selected in nonhuman primates and carefully vetted using discerning models of immune activation.
- Structure-based vector engineering. Much is known about the three-dimensional structure of AAV capsids, although less is known about capsid domains that interact with cells and proteins that drive vector performance. We plan to annotate the capsid surface with respect to the full spectrum of antibody interactions that lead to neutralization and binding to cellular proteins that drive vector efficacy and safety. This annotation will greatly inform capsid engineering efforts.
Who will conduct the work and how is it being resourced?
The work will be conducted in the Wilson laboratories at the University of Pennsylvania with the Gene Therapy Program serving as the coordinating center. Support for this program is derived exclusively from internal Penn resources and is sufficient to support a team of approximately 30 scientists.
Who owns AAV 3.0TM and will be it accessible to interested academic and commercial parties?
The work is conducted at the University of Pennsylvania, with internal resources, and therefore is owned by Penn, which controls all patenting and licensing of intellectual property that is created pursuant to this program. We plan to fully publish our work and provide open access to the promising new technology for non-commercial purposes to the academic community under standard Material Transfer Agreements. The Penn Center for Innovation is responsible for negotiating and providing commercial rights to the technology to biopharmaceutical companies and other commercial users. The overall licensing strategy will be to enable the commercial development of AAV 3.0TM as broadly as possible on commercially reasonable economic terms. More information about the Penn Center for Innovation can be found here: http://www.pci.upenn.edu/. Those wishing to learn more about the licensing opportunities for AAV 3.0TM should contact Tom Wilton at firstname.lastname@example.org.
Figure 1. The evolution of AAV vector technology. AAV 1.0 represents vectors developed from viruses isolated in the 1960s and 1970s. Vectors based on a large family of primate AAVs isolated by the Wilson Lab at the turn of the century are called AAV 2.0. The current effort by Penn to create the next-generation of AAVs led by Jim Wilson is called AAV 3.0TM.