RNA-silencing, or RNA-interference technology has recently emerged as a promising alternative to traditional gene expression inhibition and it appears to be ideally suited to effectively block the translation of specific mRNAs into their cognate proteins without the need for gene and vector transfer.

RNA-mediated gene silencing is an anti-viral evolutive cellular mechanism by virtue of which short fragments (20 to 25 base-pairs in length) of double-stranded viral RNA generated by the endonuclease Dicer are effectively used by an RNA-Induced Silencing Complex (RISC) to repeatedly identify and degrade cognate mRNA thus preventing its translation into functional cellular proteins. Similar synthetically produced double-stranded RNA oligonucleotides (generally 21 to 23 base-pair in length), known as ‘small interfering RNAs’ (siRNA), have been shown to induce the RISC to degrade complementary mRNAs in a highly selective and powerful manner. The effectiveness of this process is further enhanced by the repeat utilization of even a few siRNA molecules to achieve prolonged gene-silencing effects.

A video clip on the RNA-silencing process by siRNAs can be seen here: http://www.nature.com/focus/rnai/animations/index.html

Zyrnat Biotherapeutics has recently acquired the in-house capacity to synthesize siRNA oligonucleotides to facilitate de design and testing of experimental siRNA sequences against a variety of immune co-stimulatory and modulatory targets and further the development of its most advanced candidate, ZY-11, an anti-CD40 formulated siRNA described further below in detail.

Our lead compound, ZY-11, is a synthetic, 23 base-pair, double stranded small interfering RNA specifically designed to interfere with the synthesis of CD40 protein (TNFR5), an essential co-stimulatory molecule known to play a critical role in a variety of immune-mediated inflammatory diseases of the kidney and other organ systems through its interaction with CD40-ligand (CD40L) present on the surface of activated T-lymphocytes. The biological potency and specificity of ZY-11 have been established in a variety of in vitro and in vivo experiments where it has been shown to:

Reduce the intracellular levels of target CD40-mRNA by 85-90%.

Reduce the adherence of circulating leukocytes onto activated endothelial cells by 87%.

Appear in renal and hepatic tissues within one hour of intra-venous or intra-peritoneal administration.

Preserve renal function and reduce histological lesions in a well-established model of warm renal ischemia/reperfusion injury.

Reduce the incidence of humoral acute rejection after a single ex vivo exposure of the transplanted kidney to ZY-11 in an experimental model of renal allo-transplantation.

Preserve renal function and stall the progression of proteinuria as effectively as cyclophosphamide in an experimental in vivo model of autoimmune nephritis.

Zyrnat Biotherapeutics holds a broad European patent on ZY-11 and has filed additional global indication patents for prophylaxis of Delayed Graft Function and Acute Renal Transplant Rejection and for the treatment of Lupus Nephritis.

ZY-11 is ready to be advanced into pre-clinical regulatory development with a view to conduct clinical proof-of-principle studies in Lupus Nephritis in the near future. The cost of prescription drugs alone used in the treatment of Lupus Nephritis is estimated at $5 to 7.5 billion annually.

ZY-11 STRUCTURE & CHARACTERISTICS

Biological target: human CD40-mRNA

21 nucleotide double-stranded RNA

2 nucleotide overhang 3’ on the anti-sense strand

Chemically stabilized with partial phosphorothioate backbone and 2’-O-methyl sugar modification on the sense and antisense strands

Cholesterol conjugate to the 3’ end of the sense strand by means of a pyrrolidine linker

REFERENCES

R Pluvinet, J Pétriz, J Torras, I Herrero-Fresneda, J Torras, JM Cruzado, JM Grinyó, JM Aran. RNAi-mediated silencing of CD40 prevents leukocyte adhesion on CD154-activated endothelial cells. Blood. 2004;104:3642-3646.

R Pluvinet, R Olivar, J Krupinski, I Herrero-Fresneda, A Luque, J Torras, JM Cruzado, JM Grinyó, L Sumoy, JM Aran. CD40: an upstream master switch for endothelial cell activation uncovered by RNAi-coupled transcriptional profiling. Blood. 2008;112:3624-3637.

E Ripoll, R Pluvinet, J Torras, R Olivar, A Vidal, M Franquesa, L Cassis, JM Cruzado, O Bestard, JM Grinyó, JM Aran, I Herrero-Fresneda. In vivo therapeutic efficacy of intra-renal CD40 silencing in a model of humoral acute rejection. Gene Therapy. 2011;18:945-952.

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