Recent successes for gene therapy have targeted more readily accessible tissues, such as the liver for hemophilia (Wu et al., 2008), the eye for Leber's Congenital Amaurosis (Weber et al., 2003) and hematopoetic stem cells for SCID and adrenoleukodystrophy (Gong et al., 2015). Approaches targeting multi-organ systems or complex and inaccessible systems, such as the central nervous system (CNS), have been quite challenging using the past repertoire of gene transfer vectors. However, recent advancements in vector design technology are producing an increasing number of novel vectors that are capable of specifically, efficiently, and broadly targeting complex systems such as the CNS. The utility of such vectors is clear, since diseases of the CNS are the largest economic burden in the US. These disorders extract an enormous financial and personal toll in the United States. In 2014, it is estimated that Alzheimer's disease (AD) alone will cost $214 billion, making it the most expensive condition in the nation (Akushevich, Kravchenko, Ukraintseva, Arbeev, & Yashin, 2013). Previously, the multifaceted nature of this disease, along with inadequate vector technology, prompted a focus on small molecule treatments. Fortunately, molecular biological techniques, such as directed evolution, have provided an opportunity to develop gene therapy vectors that have the ability to target specific cell populations within the CNS. Efforts are underway by multiple groups to develop an expanding toolkit of novel gene transfer vectors, creating a need for robust high throughput and standardized methodologies with which to screen and compare these emerging vectors. This proposal will develop this essential service by producing an optical imaging method that is automated, scalable and quantitative. We leverage the use of our commercial product the TissueCyte 1000 to allow for a contract research organization (CRO) service that efficiently evaluates the bio-distribution of a potential new gene therapy vector. Here, we propose to develop an end-to-end service, in which the company is provided a high tighter virus, which we inject, measure and report back the results based on known stereotaxic axis. In addition, we will develop the requisite hardware to collect the biological material and queue the samples for analysis by PCR based strategies. The proposed service will require limited observation and intervention; thus producing highly reproducible and quantitative results. The automated processes also mean that we can quickly scale the service by the addition of more hardware, which allows the workflow to become self- sustaining. We have put together a world class team of experts from TissueVision and the University of North Carolina at Chapel Hill. Dr. Gray from UNC is a world-leading expert in the development of novel AAV vectors for CNS- directed gene therapy. He has recently published several papers (Gray et al., 2010; Gray, Matagne, et al., 2011b; Gray, Nagabhushan Kalburgi, McCown, & Jude Samulski, 2013; Kantor, Bailey, Wimberly, Kalburgi, & Gray, 2014; Powell, Rivera-Soto, & Gray, 2015) focused on the development of novel vector designs for CNS gene transfer. Dr. Gray works in close collaboration with companies such as Asklepios Biopharmaceuticals and with the UNC Vector Core facility (See Letters of Support). These groups are very interested in the service described in this proposal. Dr. Schwartz is a leader in neurobiology, data analysis, and optical imaging. Dr. Schwartz has strong experience with the pharmaceutical industry, which will be leveraged to ensure the service is amenable to a wide area of biotechnology companies (Schwartz, Piston, & DeFelice, 2006) (Gray, Foti, et al., 2011a). In conjunction with the clear positive track record from TissueVision, the association of these teams provides an ideal cohort to develop this essential service. Finally, while our emphasis is on gene therapy, the instrument and service we will build will impact a range of biomedical areas including cancer, cardiac biology, and pharmaceutical development, thus furthering its commercial appeal.
We propose to develop a service providing quantitative measures of gene transfer vector transduction efficiency. Gene therapy has seen recent resurgence in the development of specialized vectors for research or therapeutic purposes. Controlled spatial distribution and expression from gene transfer vectors is essential information regarding the utility of a viable therapeutic for gene therapy. Here, we outline an automated methodology to provide quantitative cellular content for transgene expression from various viral vectors. The successful completion of this proposal will provide a valuable resource to all investigators developing gene transfer vectors, as well as other related fields.
