The Influence of Murine Genetic Background in Adeno-Associated Virus Transduction of the Mouse Brain.

Generation of Novel AAV Variants by Directed Evolution for Improved CFTR Delivery to Human Ciliated Airway Epithelium

Efficient Whole-body Transduction with Trans-splicing Adeno-associated Viral Vectors

Comparison of AAV Serotypes for Gene Delivery to Dorsal Root Ganglion Neurons

DNA Shuffling of Adeno-associated Virus Yields Functionally Diverse Viral Progeny

Cationic Lipid Formulations Alter the In Vivo Tropism of AAV2/9 Vector in Lung

Biochemical, Pathological, and Skeletal Improvement of Mucopolysaccharidosis VI After Gene Transfer to Liver but Not to Muscle

Self-complementary AAV Vectors; Advances and Applications

Proteasome Inhibitors Enhance Gene Delivery by AAV Virus Vectors Expressing Large Genomes in Hemophilia Mouse and Dog Models: A Strategy for Broad Clinical Application

Retinal gene transfer with adeno-associated viral (AAV) vectors holds great promise for the treatment of inherited retinal degenerations (IRDs). One limit of AAV is its transfer capacity of about 5 kb, which can be expanded to about 9 kb, using dual AAV vectors. This strategy would still not suffice for treatment of IRDs such as Usher syndrome type 1D or Alström syndrome type I (ALMS) due to mutations in CDH23 or ALMS1, respectively. To overcome this limitation, we generated triple AAV vectors, with a maximal transfer capacity of about 14 kb. Transcriptomic analysis following triple AAV transduction showed the expected full-length products along a number of aberrant transcripts. However, only the full-length transcripts are efficiently translated in vivo. We additionally showed that approximately 4% of mouse photoreceptors are transduced by triple AAV vectors and showed correct localization of recombinant ALMS1. The low-photoreceptor transduction levels might justify the modest and transient improvement we observe in the retina of a mouse model of ALMS. However, the levels of transduction mediated by triple AAV vectors in pig retina reached 40% of those observed with single vectors, and this bodes well for further improving the efficiency of triple AAV vectors in the retina.

Adeno-associated Virus of a Single-polarity DNA Genome Is Capable of Transduction In Vivo

A Single Intravenous Injection of Adeno-associated Virus Serotype-9 Leads to Whole Body Skeletal Muscle Transduction in Dogs

Comparative Study of Anti-hepatitis B Virus RNA Interference by Double-stranded Adeno-associated Virus Serotypes 7, 8, and 9

INTRODUCTION. The creation of synthetic adeno-associated virus (AAV) vectors during gene therapy development is a labour-intensive and expensive process. The optimal solution to minimise the time and costs associated with gene therapy development lies in the improvement of methods aimed at assessing AAV vector biodistribution and transduction efficiency in vivo.AIM. This study aimed to develop a new bioinformatics-based assessment method for synthetic AAV vector libraries to analyse AAV vector biodistribution and transduction efficiency in vivo.MATERIALS AND METHODS. The production of synthetic AAV vectors involved assigning AAV serotype-specific barcodes (12-nucleotide tags flanked at the 5' end with a sequence encoding the green fluorescent reporter protein). Plasmids carrying unique barcodes were propagated in competent Escherichia coli XL10-Gold cells and used to create two AAV libraries: L1 with a viral genome count of 1010 and L2 with a viral genome count of 1011. AAV production involved HEK293T cell transfection. L1 and L2 library vectors were administered to C57Bl/6N mice by intravenous injection. DNA and RNA were isolated from transduced organs for analysis by next-generation sequencing. The obtained data on DNA and RNA barcode quantities in different murine organs were analysed to assess the biodistribution and transduction efficiency of synthetic AAVs. Barcodes were identified by aligning them to the expected sequences and counted. The resulting values were normalised to the quantity of barcodes in the original library.RESULTS. Seven viral constructs based on different AAV serotypes were created as part of two AAV libraries. Six of the AAV serotypes were synthetic (sAAV1, sAAV2, sAAV3, sAAV4, sAAV5, and sAAV6). Sequencing of murine organ samples revealed significant quantities of DNA barcodes from both AAV libraries in all organs except the brain. For the L1 library, RNA barcodes were detected at a sufficient level in 4 organs, including the skeletal muscles, the heart, the liver, and the adrenal glands. For the L2 library, in addition to the listed organs, sufficient RNA-barcode levels were observed in the gonads and the kidneys. According to transduction efficiency analysis based on RNA barcode levels adjusted for DNA barcodes, sAAV5 was considered the most promising variant for gene therapy of liver-related diseases, whereas sAAV2 and sAAV6 were recognised as holding the most promise for adrenal diseases.CONCLUSIONS. The developed bioinformatics-based assessment method for synthetic AAV vector libraries can analyse AAV vector biodistribution and transduction efficiency in the body. The presented approach has the potential for selecting optimal AAV vectors for specific organs and tissues in further gene therapy development.

Capsid-specific T-cell Responses to Natural Infections With Adeno-associated Viruses in Humans Differ From Those of Nonhuman Primates

Prolonged Susceptibility to Antibody-mediated Neutralization for Adeno-associated Vectors Targeted to the Liver