IsoTag™AAV: an innovative, scalable & non-chromatographic method for streamlined AAV manufacturing.

Demand for gene therapies capable of treating previously inaccessible targets has risen precipitously in the past decade. Adeno-associated viruses (AAVs) are the preferred vector for gene delivery because of their favorable safety profile and tissue tropism, but they have significant manufacturing challenges, with end-to-end yields as low as 10-30%. To combat these low yields, we developed IsoTag™AAV, a novel purification technology for AAV that is a departure from the chromatographic paradigm in downstream processing. This proprietary technology uses a self-scaffolding recombinant protein reagent that can improve manufacturing yields. It enables purification by cost-effective and scalable filtration processes and improves product quality with minimal optimization. Herein, we describe the development of IsoTag™AAV, provide a head-to-head comparison to industry-leading affinity chromatography (evaluation carried out through a joint research project with Capsida Biotherapeutics), and demonstrate how it can reduce cost of goods for a clinical AAV program by 25%.

Active Targeting of Cancer Cells by Nanobody Decorated Polypeptide Micelle with Bio-orthogonally Conjugated Drug.

Polypeptides are promising carriers for chemotherapeutics: they have minimal toxicity, can be recombinantly synthesized with precise control over molecular weight, and enhance drug pharmacokinetics as self-assembled nanoparticles. Polypeptide-based systems also provide the ability to achieve active targeting with genetically encoded targeting ligands. While passive targeting promotes accumulation of nanocarriers in solid tumors, active targeting provides an additional layer of tunable control and widens the therapeutic window. However, fusion of most targeting proteins to polypeptide carriers exposes the limitations of this approach: the residues that are used for drug attachment are also promiscuously distributed on protein surfaces. We present here a universal methodology to solve this problem by the site-specific attachment of extrinsic moieties to polypeptide drug delivery systems without cross-reactivity to fused targeting domains. We incorporate an unnatural amino acid, p-acetylphenylalanine, to provide a biorthogonal ketone for attachment of doxorubicin in the presence of reactive amino acids in a nanobody-targeted, elastin-like polypeptide nanoparticle. These nanoparticles exhibit significantly greater cytotoxicity than nontargeted controls in multiple cancer cell lines.

Heteromultivalent liposomal nanoconstructs for enhanced targeting and shear-stable binding to active platelets for site-selective vascular drug delivery.

Activated platelets play multiple roles in vascular diseases. Hence, a delivery vehicle that can specifically target activated platelets and stay retained under a hemodynamic environment can potentially enhance the efficacy of vehicle-encapsulated vascular drug by protecting it from rapid plasma deactivation, reducing its systemic non-specific side-effects, and increasing its therapeutic index at disease sites undergoing platelet hyperactivity. We rationalized that liposomal nanoconstructs surface-modified with two kinds of peptide that simultaneously bind integrin α(IIb)ß(3) and P-selectin on activated platelets can provide synergistic mechanisms for enhanced selectivity to vascular disease sites. We further hypothesized that dual-receptor targeting will enhance binding strength and retention under flow. We tested this using fluorescently-labeled liposomes, surface-modified by an RGD peptide targeted to active α(IIb)ß(3) and an EWVDV peptide targeted to P-selectin. The liposomes were studied for their platelet-specific interactions inside a parallel plate flow chamber at low-to-high shear stresses. The interaction specificity was further confirmed by flow cytometry. Our results indicate that liposomes surface-modified with both RGD and EWVDV simultaneously have higher selectivity as well as retention to activated platelets under flow compared to liposomes bearing any one peptide type. These results establish the potential of our nanoconstructs for enhanced site-selective drug delivery in vascular diseases.