ONLY A SMALL FRACTION OF CELLS PRODUCE ASSEMBLED CAPSIDS DURING TRANSFECTION-BASED MANUFACTURING OF ADENO-ASSOCIATED VIRUS VECTORS

Plasmid transfection of mammalian cells is the dominant platform used to produce adeno-associated virus (AAV) vectors for clinical and research applications. Low yields from this platform currently make it difficult to supply these activities with adequate material. In an effort to better understand the current limitations of transfection-based manufacturing, this study examines what proportion of cells in a model transfection produce appreciable amounts of assembled AAV capsid. Using conformation-specific antibody staining and flow cytometry we report the surprising result that despite obtaining high transfection efficiencies and nominal vector yields in our model system, only 5-10% of cells appear to produce measurable levels of assembled AAV capsids. This finding implies that considerable increases in vector titer could be realized through increasing the proportion of productive cells. Furthermore, we suggest that the flow cytometry assay used here to quantify productive cells may be a useful metric for future optimization of transfection-based AAV vector manufacturing platforms.

146 Evolving Mutliplexed shRNA to generate tailored CAR T cell therapy

BackgroundManipulating protein expression to generate cells with a specific desired phenotype is one of the central goals of engineered cell therapy. Short Hairpin RNA (shRNA) is a well-established approach to reduce protein expression through the targeted degradation of messenger RNA transcripts. However, the use of shRNA in the Chimeric Antigen Receptor (CAR) T cell therapy has been limited. We have recently shown that single shRNA incorporated into a CAR expression vector can knockdown expression of the target antigen when expressed on the CAR T itself to avoid fratricide or expression of some elements of the T cell receptor (TCR) to generate allogeneic CAR T cells. An attraction of the shRNA approach is to express multiple shRNA from the same vector that can regulate protein expression thereby optimzing CAR T cell phenotype.MethodsRetroviral vectors encoding a CAR targeting a well-studied antigen (generally BCMA) co-expressing a tag for cell enrichment and identification along with shRNA multiplexed were generated. The shRNA multiplexed were inserted within a microRNA (miR) framework to enable expression from a single PolII promoter (the retroviral LTR promoter). Functional assessment of target knockdown target in T cells along with retroviral titers was determinedResultsOur products in ongoing clinical development have employed a miR196a2 scaffold enabling the expression of the desired shRNA driven by the same promoter as that used for the CAR and other transgenes. Multiplexing the miR196a2 scaffold to express multiple shRNA (targeting CD247, beta 2 Microglobulin and CD95) was successful in terms of target knockdown but an obvious reduction in retroviral titer was observed. These titer reductions were variable between the duplex and triplex shRNA constructs examined but were uniformly low when considering clinical development. A proprietary scaffold was developed that coupled expression of duplexed and triplexed shRNA while also elevating vector titer by at least 2-3x.ConclusionsMultiplexing shRNA within a single vector format with scaffolds that ensure co-linked expression of the shRNA with therapeutic transgenes is a highly attractive approach to generate CAR T cells with bespoke, desired phenotypes. However, simply multiplexing shRNA using a currently clinical-used scaffold (miR196a2) resulted in reductions in vector titer. Engineering further proprietary scaffolds were produced that maintained shRNA expression but elevated retroviral titer to a level which does not preclude clinical development. These developments now provide the opportunity to develop second generation clinical candidates using shRNA multiplexed technology.

Controlled Release of rAAV Vectors from APMA-Functionalized Contact Lenses for Corneal Gene Therapy

As an alternative to eye drops and ocular injections for gene therapy, the aim of this work was to design for the first time hydrogel contact lenses that can act as platforms for the controlled delivery of viral vectors (recombinant adeno-associated virus, rAAV) to the eye in an effective way with improved patient compliance. Hydrogels of hydroxyethyl methacrylate (HEMA) with aminopropyl methacrylamide (APMA) (H1: 40, and H2: 80 mM) or without (Hc: 0 mM) were synthesized, sterilized by steam heat (121 °C, 20 min), and then tested for gene therapy using rAAV vectors to deliver the genes to the cornea. The hydrogels showed adequate light transparency, oxygen permeability, and swelling for use as contact lenses. Loading of viral vectors (rAAV-lacZ, rAAV-RFP, or rAAV-hIGF-I) was carried out at 4 °C to maintain viral vector titer. Release in culture medium was monitored by fluorescence with Cy3-rAAV-lacZ and AAV Titration ELISA. Transduction efficacy was tested through reporter genes lacZ and RFP in human bone marrow derived mesenchymal stem cells (hMSCs). lacZ was detected with X-Gal staining and quantified with Beta-Glo®, and RFP was monitored by fluorescence. The ability of rAAV-hIGF-I-loaded hydrogels to trigger cell proliferation in hMSCs was evaluated by immunohistochemistry. Finally, the ability of rAAV-lacZ-loaded hydrogels to transduce bovine cornea was confirmed through detection with X-Gal staining of β-galactosidase expressed within the tissue.