In vivo gene replacement for the treatment of inherited disease is one of the most compelling concepts in modern medicine. Adeno-associated virus (AAV) vectors have been extensively used for this purpose and have shown therapeutic efficacy in a range of animal models. Successful translation to the clinic was initially slow, but long-term expression of donated genes at therapeutic levels has now been achieved in patients with inherited retinal disorders and haemophilia B. Recent exciting results have raised hopes for the treatment of many other diseases. As we discuss here, the prospects and challenges for AAV gene therapy are to a large extent dependent on the target tissue and the specific disease.

Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges

Gene therapy is finally poised to make a contribution to the treatment of debilitating, highly penetrant genetic diseases that have proved intractable to other regimens.

Gene therapy finds its niche

Comparative analyses of adeno-associated viral vector serotypes 1, 2, 5, 8 and 9 in marmoset, mouse and macaque cerebral cortex.

Gene therapy with adeno-associated virus vector 5-human factor IX in adults with hemophilia B.

This protocol describes the use of Cas9 ribonucleoproteins and AAV6 donor templates to enable precise genome editing of human hematopoietic stem cells by homologous recombination (HR), including in vitro and in vivo assays for determining HR frequencies in HSPCs. Genome editing via homologous recombination (HR) (gene targeting) in human hematopoietic stem cells (HSCs) has the power to reveal gene–function relationships and potentially transform curative hematological gene and cell therapies. However, there are no comprehensive and reproducible protocols for targeting HSCs for HR. Herein, we provide a detailed protocol for the production, enrichment, and in vitro and in vivo analyses of HR-targeted HSCs by combining CRISPR/Cas9 technology with the use of rAAV6 and flow cytometry. Using this protocol, researchers can introduce single-nucleotide changes into the genome or longer gene cassettes with the precision of genome editing. Along with our troubleshooting and optimization guidelines, researchers can use this protocol to streamline HSC genome editing at any locus of interest. The in vitro HSC-targeting protocol and analyses can be completed in 3 weeks, and the long-term in vivo HSC engraftment analyses in immunodeficient mice can be achieved in 16 weeks. This protocol enables manipulation of genes for investigation of gene functions during hematopoiesis, as well as for the correction of genetic mutations in HSC transplantation–based therapies for diseases such as sickle cell disease, β-thalassemia, and primary immunodeficiencies.

CRISPR/Cas9 genome editing in human hematopoietic stem cells

A recombinant adeno-associated virus serotype 2 Reference Standard Material (rAAV2 RSM) has been produced and characterized with the purpose of providing a reference standard for particle titer, vector genome titer, and infectious titer for AAV2 gene transfer vectors Production and purification of the reference material were carried out by helper virus-free transient transfection and chromatographic purification The purified bulk material was vialed, confirmed negative for microbial contamination, and then distributed for characterization along with standard assay protocols and assay reagents to 16 laboratories worldwide Using statistical transformation and modeling of the raw data, mean titers and confidence intervals were determined for capsid particles ({X}, 918 x 10¹¹ particles/ml; 95% confidence interval [CI], 789 x 10¹¹ to 105 x 10¹² particles/ml), vector genomes ({X}, 328 x 10¹⁰ vector genomes/ml; 95% CI, 270 x 10¹⁰ to 475 x 10¹⁰ vector genomes/ml), transducing units ({X}, 509 x 10⁸ transducing units/ml; 95% CI, 200 x 10⁸ to 960 x 10⁸ transducing units/ml), and infectious units ({X}, 437 x 10⁹ TCID₅₀ IU/ml; 95% CI, 206 x 10⁹ to 926 x 10⁹ TCID₅₀ IU/ml) Further analysis confirmed the identity of the reference material as AAV2 and the purity relative to nonvector proteins as greater than 94% One obvious trend in the quantitative data was the degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution This relatively poor degree of interlaboratory precision and accuracy was apparent even though attempts were made to standardize the assays by providing detailed protocols and common reagents This is the first time that such variation between laboratories has been thoroughly documented and the findings emphasize the need in the field for universal reference standards The rAAV2 RSM has been deposited with the American Type Culture Collection and is available to the scientific community to calibrate laboratory-specific internal titer standards Anticipated uses of the rAAV2 RSM are discussed

http://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=1051&context=peds_pulmonary

Tina Philipsberg

Papers

Characterization of a recombinant adeno-associated virus type 2 Reference Standard Material.