Genome length determination in adeno-associated virus vectors with mass photometry.

Recombinant adeno-associated viruses (rAAVs) are attractive therapeutic viral vectors for gene delivery. To ensure the efficacy and safety of rAAV-based therapies, comprehensive characterization of the adeno-associated virus (AAV) capsids is essential. Mass photometry (MP) provides the advantage of short analysis times, low sample consumption, and high accuracy of molecular mass determination. Despite having just recently emerged, MP has already been used to characterize AAV genome content and quantify filled/empty capsid ratios. In this study, we explored three approaches for the application of MP to assess genome length in AAVs. In approach 1, genome length in intact AAVs was approximated with good precision (coefficient of variation [%CV] < 2.6%) and accuracy (±5%) by using a straightforward protein-based calibration. In approach 2, genome length was determined even more accurately (±1%, %CV < 2.9%) considering calibration with a set of additional AAVs of different genome length. In approach 3, genome length was assessed after genome release from the capsid by heating in 1% sodium dodecyl sulfate followed by surfactant removal with precision of %CV < 0.7% and accuracy of ±5%. In conclusion, the three developed MP-based approaches are fast, precise, and accurate methods for genome length determination in AAVs, differing in their calibration materials and efforts.

A Bioengineered In Vitro Model to Assess AAV-Based Gene Therapies for Cyclic GMP-Related Disorders.

The emergence of efficient viral vectors derived from adeno-associated viruses (AAV) has led many groups to develop gene therapies for inherited monogenic diseases, such as retinal dystrophies. To evaluate the potency of new gene therapy vectors in a preclinical context, it is common to use animal models, such as gene-deficient or mutant animal models of a given human disease, and then assess vision restoration with functional or behavioral assays. While such animal models are invaluable to the preclinical testing process, they cannot be readily used as batch release tests during manufacturing or to validate biological activity at later stages of development. There is therefore a need for rapid and reliable in vitro models that can determine whether therapeutic vectors have delivered their cargo gene, and more importantly, whether this has resulted in the intended biological activity. Given our previous experience, we chose CNGA3-linked achromatopsia to develop a cell-based system to verify biological activity of AAV vectors designed to deliver a healthy CNGA3 gene copy into human cone photoreceptors. Our system is based on an immortalized cell line with high susceptibility to AAV transduction, i.e., HeLa cells, which we engineered to express a fungal rhodopsin guanylyl cyclase (RhGC) from Blastocladiella emersonii and a sensitive genetically encoded calcium indicator (GECI) under the control of a tetracycline operator. Using this system, we were able to confirm and quantify the function of the ion channel encoded by AAV/CNGA3 and differentiate between AAV vector potencies with a simple fluorometric assay. Finally, we show that this approach can be readily adapted for the assessment of phosphodiesterase function.

Novel AAV capsids for intravitreal gene therapy of photoreceptor disorders.

Gene therapy using recombinant adeno-associated virus (rAAV) vectors to treat blinding retinal dystrophies has become clinical reality. Therapeutically impactful targeting of photoreceptors still relies on subretinal vector delivery, which detaches the retina and harbours substantial risks of collateral damage, often without achieving widespread photoreceptor transduction. Herein, we report the development of novel engineered rAAV vectors that enable efficient targeting of photoreceptors via less invasive intravitreal administration. A unique in vivo selection procedure was performed, where an AAV2-based peptide-display library was intravenously administered in mice, followed by isolation of vector DNA from target cells after only 24 h. This stringent selection yielded novel vectors, termed AAV2.GL and AAV2.NN, which mediate widespread and high-level retinal transduction after intravitreal injection in mice, dogs and non-human primates. Importantly, both vectors efficiently transduce photoreceptors in human retinal explant cultures. As proof-of-concept, intravitreal Cnga3 delivery using AAV2.GL lead to cone-specific expression of Cnga3 protein and rescued photopic cone responses in the Cnga3-/- mouse model of achromatopsia. These novel rAAV vectors expand the clinical applicability of gene therapy for blinding human retinal dystrophies.