Fast-Seq, a simple method for rapid and inexpensive validation of packaged ssAAV genomes in academic settings

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

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

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

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

Site-specific Modification of AAV Vector Particles With Biophysical Probes and Targeting Ligands Using Biotin Ligase

Self-complementary AAV Vectors; Advances and Applications

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

Suppression and Replacement Gene Therapy for Autosomal Dominant Disease in a Murine Model of Dominant Retinitis Pigmentosa

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.

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

Adeno-Associated Virus Vectors in Clinical Trials

Adeno-associated viral (AAV) vectors have shown great promise in gene delivery as evidenced by recent FDA approvals. Despite efforts to optimize manufacturing for good manufacturing practice (GMP) productions, few academic laboratories have the resources to assess vector composition. One critical component of vector quality is packaged genome fidelity. Errors in viral genome replication and packaging can result in the incorporation of faulty genomes with mutations, truncations, or rearrangements, compromising vector potency. Thus, sequence validation of packaged genome composition is an important quality control (QC), even in academic settings. We developed Fast-Seq, an end-to-end method for extraction, purification, sequencing, and data analysis of packaged single-stranded AAV (ssAAV) genomes intended for non-GMP preclinical environments. We validated Fast-Seq on ssAAV vectors with three different genome compositions (CAG-GFP, CAG-tdTomato, EF1α-FLuc), three different genome sizes (2.9, 3.6, 4.4 kb), packaged in four different capsid serotypes (AAV1, AAV2, AAV5, and AAV8), and produced using the two most common production methods (Baculovirus-Sf9 and human HEK293), from both common commercial vendors and academic core facilities supplying academic laboratories. We achieved an average genome coverage of >1,400 × and an average inverted terminal repeat coverage of >280 × , despite the many differences in composition of each ssAAV sample. When compared with other ssAAV next-generation sequencing (NGS) methods for GMP settings, Fast-Seq has several unique advantages: Tn5 transposase-based fragmentation rather than sonication, 125 × less input DNA, simpler adapter ligation, compatibility with commonly available inexpensive sequencing instruments, and free open-source data analysis code in a preassembled customizable Docker container designed for novices. Fast-Seq can be completed in 18 h, is more cost-effective than other NGS methods, and is more accurate than Sanger sequencing, which is generally only applied at 1–2 × sequencing depth. Fast-Seq is a rapid, simple, and inexpensive methodology to validate packaged ssAAV genomes in academic settings.

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

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

Efficient Intrathymic Gene Transfer Following In Situ Administration of a rAAV Serotype 8 Vector in Mice and Nonhuman Primates