Animal models of atherosclerosis.

RNAi therapeutic and its innovative biotechnological evolution.

Objectives— Patients with mutations in the proprotein convertase subtilisin/kexin type 9 ( PCSK9 ) gene have hypercholesterolemia and are at high risk of adverse cardiovascular events. We aimed to stably express the pathological human D374Y gain-of-function mutant form of PCSK9 ( PCSK9 DY ) in adult wild-type mice to generate a hyperlipidemic and proatherogenic animal model, achieved with a single systemic injection with adeno-associated virus (AAV). Approach and Results— We constructed an AAV-based vector to support targeted transfer of the PCSK9 DY gene to liver. After injection with 3.5×10 10 viral particles, mice in the C57BL/6J, 129/SvPasCrlf, or FVB/NCrl backgrounds developed long-term hyperlipidemia with a strong increase in serum low-density lipoprotein. Macroscopic and histological analysis showed atherosclerotic lesions in the aortas of AAV- PCSK9 DY mice fed a high-fat-diet. Advanced lesions in these high-fat-diet–fed mice also showed evidence of macrophage infiltration and fibrous cap formation. Hepatic AAV- PCSK9 DY infection did not result in liver damage or signs of immunologic response. We further tested the use of AAV- PCSK9 DY to study potential genetic interaction with the ApoE gene. Histological analysis of ApoE −/− AAV- PCSK9 DY mice showed a synergistic response to ApoE deficiency, with aortic lesions twice as extensive in ApoE −/− AAV- PCSK9 DY -transexpressing mice as in ApoE −/− AAV- Luc controls without altering serum cholesterol levels. Conclusions— Single intravenous AAV- PCSK9 DY injection is a fast, easy, and cost-effective approach, resulting in rapid and long-term sustained hyperlipidemia and atherosclerosis. We demonstrate as a proof of concept the synergy between PCSK9 DY gain-of-function and ApoE deficiency. This methodology could allow testing of the genetic interaction of several mutations without the need for complex and time-consuming backcrosses.

Induction of Sustained Hypercholesterolemia by Single Adeno-Associated Virus–Mediated Gene Transfer of Mutant hPCSK9

Recombinant AAV as a Platform for Translating the Therapeutic Potential of RNA Interference

The major impact produced by the severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) focused many researchers attention to find treatments that can suppress transmission or ameliorate the disease. Despite the very fast and large flow of scientific data on possible treatment solutions, none have yet demonstrated unequivocal clinical utility against coronavirus disease 2019 (COVID‑19). This work represents an exhaustive and critical review of all available data on potential treatments for COVID‑19, highlighting their mechanistic characteristics and the strategy development rationale. Drug repurposing, also known as drug repositioning, and target based methods are the most used strategies to advance therapeutic solutions into clinical practice. Current in silico, in vitro and in vivo evidence regarding proposed treatments are summarized providing strong support for future research efforts.

/pdf/comprehensive-analysis-of-drugs-to-treat-sars-cov-2-a94rc029gv.pdf

Comprehensive analysis of drugs to treat SARS­CoV­2 infection: Mechanistic insights into current COVID­19 therapies (Review).

https://northumbria-test.eprints-hosting.org/id/document/261949

Safe, long-term hepatic expression of anti-HCV shRNA in a nonhuman primate model

This invention is directed to an RNA interference (RNAi) agent and the use of that RNAi agent to treat Age-related Macular Degeneration, as well as pharmaceutical compositions containing the RNAi agents of the invention. The RNAi agent is a DNA-directed RNA interference (ddRNAi) agent (being an RNA molecule), together with an expression cassette or construct to express that agent in a cell (including in vivo), for inhibiting, preventing or reducing expression of an AMD associated gene. Preferably that AMD associated gene is one that is associated with wet AMD.

Age-related macular degeneration treatment

Oculopharyngeal muscular dystrophy (OPMD) is a rare autosomal dominant disease that results from an alanine expansion in the N-terminal domain of Poly-A Binding Protein Nuclear-1 (PABPN1). We have recently demonstrated that a two-vector gene therapy strategy significantly ameliorated the pathology in a mouse model of OPMD. This approach entailed intramuscular injection of two recombinant adeno-associated viruses (AAVs), one expressing three short hairpin RNAs (shRNAs) to silence both mutant and wild-type PABPN1 and one expressing a codon-optimized version of PABPN1 that is insensitive to RNA interference. Here we report the continued development of this therapeutic strategy by delivering "silence and replace" sequences in a single AAV vector named BB-301. This construct is composed of a modified AAV serotype 9 (AAV9) capsid that expresses a unique single bifunctional construct under the control of the muscle-specific Spc5-12 promoter for the co-expression of both the codon-optimized PABPN1 protein and two small inhibitory RNAs (siRNAs) against PABPN1 modeled into microRNA (miRNA) backbones. A single intramuscular injection of BB-301 results in robust inhibition of mutant PABPN1 and concomitant replacement of the codon-optimized PABPN1 protein. The treatment restores muscle strength and muscle weight to wild-type levels as well as improving other physiological hallmarks of the disease in a mouse model of OPMD.

BB-301: a silence and replace AAV-based vector for the treatment of oculopharyngeal muscular dystrophy

The present disclosure relates to modified adeno-associated virus (AAV) delivery vectors comprising 'silence and replace' DNA constructs, compositions comprising same, and the use of the modified AAV and compositions to treat oculopharyngeal muscular dystrophy (OPMD) in individuals suffering from OPMD or which are predisposed thereto. In particular, the disclosure relates to AAV having a capsid protein with a modified subunit 1 (VP1) and comprising a 'silence and replace' DNA construct, wherein the 'silence and replace' DNA construct comprises (i) a DNA-directed RNAi (ddRNAi) construct encoding short hairpin microRNA (shmiR) targeting PABPN1 causative of OPMD and (ii) a PABPN1 construct encoding functional PABPN1 protein having a mRNA transcript which is not targeted by the shmiRs at (i). The present disclosure also relates to the methods of treating OPMD comprising direct injection of an AAV of the disclosure to a subject's pharyngeal muscles.

Compositions and methods for treating oculopharyngeal muscular dystrophy (opmd)

This disclosure relates to RNA interference (RNAi) reagents for treatment or prevention of ocular diseases and conditions associated with neovascularisation, such as age-related macular degeneration (AMD), compositions comprising same, and use thereof to treat individuals suffering from, or predisposed to, ocular diseases and conditions associated with neovascularisation, such as AMD.

Shih-Chu Kao

Papers

Safe, long-term hepatic expression of anti-HCV shRNA in a nonhuman primate model

Age-related macular degeneration treatment

BB-301: a silence and replace AAV-based vector for the treatment of oculopharyngeal muscular dystrophy

Compositions and methods for treating oculopharyngeal muscular dystrophy (opmd)

Reagents for treatment of ocular diseases and conditions associated with neovascularisation and use thereof