VIC Led by

Kevin Wickman, Medical School and Daniel Schmidt, College of Biological Sciences

The Viral Innovation Core (VIC) aims to develop novel genetic approaches for the experimental study of the anatomical, cellular, and molecular bases of addiction.

P30DA048742-01A1, sub-project 6725

Contemporary investigations related to the neuroscience of addiction utilize a broad set of genetically encoded tools that can be used to control the excitability of defined neurons within circuits, highlight connectivity between neurons that form microcircuits, and report cellular activity states in behaving animals. Vectors exploiting the beneficial features of the Adeno-Associated Virus (AAV) backbone have proven invaluable for delivering genetically encoded tools to specific cell types and circuits in the nervous system. One goal of the Viral Innovation Core (VIC) is to meet the AAV vector needs of the addiction research community, providing investigators with access to advanced and experimental viral production services. A second goal of the VIC is to develop new AAV-based tools that can surmount the current limitations associated with viral tropism and expand the capacity to target neuron populations of interest in rodents, non-human primates, and other species of interest.

CURRENT SERVICES

AAV production and packaging

Working in conjunction with the University of Minnesota Viral Vector and Cloning Core (VVCC), the VIC supports the generation of AAV vectors – including custom vectors – for members of the UMN addiction research community. Providing this labor-intensive service through a centralized entity with skilled staff represents a critical efficiency for the VIC user base, and facilitates the centralized examination, evaluation, and interpretation of data from a large and broad array of vector tools. Tools generated with support from the VIC are expected to promote engagement with the other three Cores, creating synergies that will strengthen the impact and innovation of all supported projects, and enhance the scope and impact of UMN research in the area of addiction.

Projects undertaken by the VIC could include the packaging of stock/commercial AAV vectors including those that express genetically encoded fluorescent proteins (e.g., GFP, mCherry) and/or neuromodulatory tools (opsins, DREADDs) under the control of cell-specific promoters (e.g., hSyn, CaMKIIα). These and other vectors can be packaged in a serotype (e.g., AAV1, 2, 3, 4 5, 6, 7, 8, 9, rh10, AAV2retro, DJ, PHP.B, PHPeB, and PHP.S) suitable for the cell population under investigation. AAV vectors are concentrated and purified by sucrose cushion ultracentrifugation, and viral titer is determined by digital PCR. The typical yield is 200 uL of AAV vector (>1x10¹³ genocopies/mL). Vectors produced by the VIC have proven to be of sufficient quality and purity for use in the most sensitive in vitro (e.g., primary cell cultures) or in vivo (e.g., intracranial manipulations) applications.

The VIC will also support the development of custom reagents to fill project-specific needs, including vectors harboring gene-specific shRNA or guide RNAs, or cDNAs downstream from a promoter of interest. Cre-dependent (DIO and DO) versions of AAV vectors are popular among VIC clients, as they can be used in conjunction with an expanding array of Cre transgenic mouse and rat lines that have revolutionized circuit-based neuroscience investigations over the last decade. VIC staff will design and produce the DNA plasmids required for custom AAV production.

Optimizing viral tropism

For most research studies involving viral vectors, it is desirable to restrict manipulations to a specific cell type. Cell specificity with viral manipulations is currently achieved with DNA regulatory elements such as cell-specific promoters or via transgenic animals expressing Cre or Flp recombinase in specific cell types. While cell-specific promoters are widely used, these promoters only coarsely segregate cell types. In addition, it can be difficult to balance cell specificity with strength of expression when the expressed transgene is toxic at high expression levels. Furthermore, intersectional approaches (e.g., pairing expression of Cre recombinase driven by one promoter with a “floxed” transgene whose expression is driven by another promoter) only work with tightly regulated promoters, as even leaky expression of recombinase can be sufficient for transgene recombination. While transgenic driver lines can facilitate neuron-specific viral manipulations, this approach is expensive, is largely restricted to mice, and is unlikely to be broadly available in more translationally relevant non-human primate (NHP) models. Accordingly, the VIC is also pursuing innovative solutions to the tropism-related limitations of current AAV vectors. New multi-modal AAV targeting paradigms will represent a substantial benefit to the broader scientific research community.

The VIC directs a research and development component with the goal of improving performance and utility of AAV vectors, oriented around the specific needs of the UMN addiction research community. The VIC is comprehensively and quantitatively mapping the engineerable potential of capsids of several AAV serotypes. This is helping us define capsid regions that are permissive to insertion of a protein domain that mediates programmable and covalent attachment of antibodies or other non-immunoglobulin scaffolds, which recognize surface receptors on addiction-relevant cell populations. ‘Arming’ capsids in this fashion thus redirects AAV tropism in a user-defined and predictable manner. Receptor-mediated gene delivery represents a powerful approach to achieving precise manipulation of neural circuits.

SERVICES COMING SOON

Advanced viral vector characterization and performance tracking. All vectors generated with VIC support will undergo a rigorous characterization process that will shed light on the purity, true infective titer, and stability of the reagent. The advanced vector characterization approach will help to establish best practices related to viral production and purification, which will improve the likelihood of success and the reliability of all supported research projects.

The VIC will employ a stringent process of product evaluation and quality control that will involve: a) screening for contaminating host cell DNA, host cell protein, and encapsidated helper component DNA, b) determining the ratio of empty capsid and encapsidated genomes, and c) measuring the stability of virus capsid. In aggregate, these data will represent a comprehensive profile of virus quality that can be used to help optimize vector production and purification approaches. Moreover, as capsid stability and dynamics could influence key aspects of vector function, including tropism, shelf life, and transduction efficiency, routine acquisition of these data – combined with application-specific feedback – will help the VIC best advise Investigators related to the design, use, and storage of these tools.

Over time, and with feedback from Investigators on AAV vector performance in project-specific settings,, data generated via this vector production and advanced characterization process will guide decision-making regarding subsequent vector design and production, and increase the likelihood that data obtained using AAV vectors will be reproducible.

VIC Staff

Kevin Wickman, PhD. Dr. Wickman is a Professor and Head of Pharmacology, and he established the UMN Viral Vector and Cloning Core. Dr. Wickman coordinates the activities of the VIC and VVCC to ensure that the UMN addiction research community has access to high-quality AAV vectors to support their independent research programs.

Daniel Schmidt, PhD. Dr. Daniel Schmidt is an Assistant Professor in the UMN Genetics, Cell Biology, and Development Department, and he has directed a BRAIN Initiative-funded viral tool development project. He is leveraging his expertise in the area of innovative viral tool and delivery development to provide effective guidance to Investigators interested in utilizing AAV vectors in their research, and for developing new approaches to addressing the limitations of AAV vector tropism.

Ezequiel Marron, PhD. Dr. Marron is the VVCC Manager, and he serves as a critical liaison between the VVCC and VIC. Dr. Marron has cultivated a strong relationship with the UMN neuroscience research community by virtue of his many years of practical experience with the application of viral technologies to neuroscience-related research questions, and his strong commitment to facilitating research at the UMN. Dr. Marron oversees custom vector design and coordinates the efforts of VVCC cloning and packaging specialists to ensure that client projects are completed in a timely and accurate manner.

Mehrsa Zahiremami. Mehrsa is an AAV packaging specialist responsible for the cell-based production and purification of high-titer AAV vectors.

Melody Truong. Melody is a DNA cloning specialist responsible for completing custom cloning projects required for custom AAV vector production.

Tyler Haeberle. Tyler is a DNA cloning specialist responsible for completing custom cloning projects required for custom AAV vector production.

Publications