Graduate Student, California Institute of Technology
Development of novel gene delivery viral vectors to target specific cell type or tissue of interest.
The use of gene delivery vehicles to target a tissue or a cell-type with greater specificity and efficiency is a primary need for any gene therapy or gene perturbation studies. There are a few viral vectors which have been recognized for these purposes. A promising candidate among them is the recombinant adeno-associated virus (rAAV), a non-enveloped, single stranded DNA virus with a capsid protein coat. Their non-pathogenicity and ability to infect a wide range of diving and non-diving cells (nerve cells) makes them appealing for applications in basic science research and in gene therapy trials for genetic diseases. However, there is a need to engineer these vectors for our specific needs. Currently the use of rAAV suffers from low transduction or infection efficiency and broad tropism which makes it difficult to target gene delivery to a specific region. Several groups have tried to design novel rAAVs using rational designs or directed evolution approach. The evolution method is more promising when we have little understanding of how the viral tropism works. We can easily build a large library (several thousand) of novel rAAVs and allow natural selection to help us narrow down on the variants that performed the best. Recently, one such selection method called CREATE was published in Nature Biotechnology 2016 from Dr. Viviana Gradinaru’s laboratory at Caltech. In this paper, we have developed a novel rAAV capsid called AAV-PHP.B that was evolved from a wild-type rAAV9. This variant transduces the central nervous system (CNS) with 40 fold higher efficiency compared to the parent. This was achieved using a “cre recombination-based AAV targeted evolution method” (CREATE) where the variants were selectively recovered from the nerve cell astrocytes of genetically modified mice. In 2017, few more variants were reported in Nature Neuroscience from the same laboratory using the same method. One variant is specific for sensory neurons (AAV-PHP.S) and another has enhanced broad CNS transduction with higher specificity to neurons (AAV-PHP.eB). My work focuses on finding novel variants targeting different nerve cell types that were not studied earlier such as microglia and interneurons. These nerve cell types are hard to target and will be very useful tool for the scientific community. In addition, my work focuses on finding novel variants targeting different organs such as heart, lung, small intestine and muscle which are widely studied for genetic diseases.
Abstract: Adeno-associated viral (AAV) vectors have shown promise as a platform for gene therapy of neurological disorders. Achieving global gene delivery to the central nervous system (CNS) is key for development of effective therapies for many of these diseases. Here we report the isolation of a novel CNS tropic AAV capsid, AAV-B1, after a single round of in vivo selection from an AAV capsid library. Systemic injection of AAV-B1 vector in adult mice and cat resulted in widespread gene transfer throughout the CNS with transduction of multiple neuronal sub-populations. In addition, AAV-B1 transduces muscle, beta cells, pulmonary alveoli and retinal vasculature at high efficiency. This vector is more efficient than AAV9 for gene delivery to mouse brain, spinal cord, muscle, pancreas and lung. Together with reduced sensitivity to neutralization by antibodies in pooled human sera, the broad transduction profile of AAV-B1 represents an important improvement over AAV9 for CNS gene therapy.Molecular Therapy (2016); doi:10.1038/mt.2016.84.
Pub.: 28 Apr '16, Pinned: 28 Jun '17
Abstract: Adeno-associated viruses (AAVs) are commonly used for in vivo gene transfer. Nevertheless, AAVs that provide efficient transduction across specific organs or cell populations are needed. Here, we describe AAV-PHP.eB and AAV-PHP.S, capsids that efficiently transduce the central and peripheral nervous systems, respectively. In the adult mouse, intravenous administration of 1 × 1011 vector genomes (vg) of AAV-PHP.eB transduced 69% of cortical and 55% of striatal neurons, while 1 × 1012 vg of AAV-PHP.S transduced 82% of dorsal root ganglion neurons, as well as cardiac and enteric neurons. The efficiency of these vectors facilitates robust cotransduction and stochastic, multicolor labeling for individual cell morphology studies. To support such efforts, we provide methods for labeling a tunable fraction of cells without compromising color diversity. Furthermore, when used with cell-type-specific promoters and enhancers, these AAVs enable efficient and targetable genetic modification of cells throughout the nervous system of transgenic and non-transgenic animals.
Pub.: 26 Jun '17, Pinned: 28 Jun '17
Abstract: Recombinant adeno-associated viruses (rAAVs) are commonly used vehicles for in vivo gene transfer1, 2, 3, 4, 5, 6. However, the tropism repertoire of naturally occurring AAVs is limited, prompting a search for novel AAV capsids with desired characteristics7, 8, 9, 10, 11, 12, 13. Here we describe a capsid selection method, called Cre recombination–based AAV targeted evolution (CREATE), that enables the development of AAV capsids that more efficiently transduce defined Cre-expressing cell populations in vivo. We use CREATE to generate AAV variants that efficiently and widely transduce the adult mouse central nervous system (CNS) after intravenous injection. One variant, AAV-PHP.B, transfers genes throughout the CNS with an efficiency that is at least 40-fold greater than that of the current standard, AAV9 (refs. 14,15,16,17), and transduces the majority of astrocytes and neurons across multiple CNS regions. In vitro, it transduces human neurons and astrocytes more efficiently than does AAV9, demonstrating the potential of CREATE to produce customized AAV vectors for biomedical applications.
Pub.: 01 Feb '16, Pinned: 28 Jun '17