Recombinant AAV vectors have shown great promise in clinical trials. These vectors represent a gene transfer/genome editing platform that has the potential to treat not only genetic diseases but a myriad of acquired disorders that include infection and infection prophylaxis, neurodegeneration, and diseases resulting from immune system dysfunction. One of the major rate-limiting steps in translating the success achieved in animal models of human disease to humans is the lack of a strong correlation between vector transduction properties between species. Because transduction is dictated in large part by variations in the capsid protein sequence, in order to obtain capsids with enhanced transducing properties in humans we have pursued multi- species capsid shuffling, and in vitro and in vivo evolutionary selection paradigms to create and identify novel chimeric capsids with clinically relevant assets. During the current funding period, we discovered several chimeric capsids with a 10-fold increased primate liver transduction profile. One of these capsids is in clinical trials, and two more recent isolates are in late preclinical testing by commercial and academic centers. Yet even these improved AAV vectors do not appear to reach the same level of transduction that can be achieved in rodents with other established AAV capsids. Thus, the general goal of the proposed work is to build upon our efforts to develop high throughput technologies for new capsid engineering approaches, and optimized selection schemes. Our specific goals are to create and identify capsids that have enhanced: (1) human liver transduction, (2) penetration through the human blood brain barrier and transduction of neurons and astrocytes, and (3)transduction of human hematopoietic stem cells for increased genome editing efficiencies. We will also study the mechanism behind the species selectivity observed with several of our new specific chimeric capsid derived vectors. The vectors that are obtained in the respective screens will be further evaluated in either an appropriate humanized animal model or non-human primates. The information learned will contribute to our knowledge towards optimizing AAV-mediated gene transfer in humans. The new capsids will be made available for use in clinical gene transfer/genome editing trials.