ddRNAI A direct shot treatment that they are carefully...

ddRNAI   A direct shot treatment that they are carefully progressing in human trials far superior to current SIRNA which  has to be synthesized externally  and introduced into  the cell that is time consuming to treat and still being perfected in humans
Refer to benitec website
SiRNA or shRNA?

Whilst there are several options for synthetic and expressed RNAi: the most commonly-used form of synthetic RNAi is siRNA, and of expressed RNAi is shRNA. Benitec's ddRNAi patents cover the use of all forms of expressed RNAi in humans, including shRNA
siRNA (small interfering RNA), is an intermediate in the RNA interference (RNAi) pathway. It occurs naturally in the cytoplasm or may be synthesized externally and introduced to the cell.
shRNA (short hairpin RNA) is the most widely used form of ddRNAi. shRNA is produced inside the target cell from a DNA construct that has been delivered to the nucleus.

The differences between them are summarized in this table, based on Rao, Vorhies, Senzer et al¹ and DiGiusto, Krishnan, Li et al² and described in more detail below.

	Column 1	Column 2	Column 3
0	Criterion	siRNA	shRNA
1	Nomenclature	Small Interfering RNA	Short Hairpin RNA
2	Source	Laboratory synthesis	Nuclear expression
3	Delivery to the cell	Via synthetic/natural polymers and lipids to the cytoplasm	Via viral and other gene therapy vectors to the nucleus.
4	Persistence	99% degraded after 48 hours	Expressed for up to 3 years.
5	Administration	Local or limited systemic injection	Local and systemic injection
6	Dosage	High (low nM)	Low (5 copies)
7	Likelihood of specific ‘off target’ effects	Higher than shRNA	Lower than siRNA
8	Likelihood of non-specific ‘off targets’ effects	Higher immune activation, inflammation and toxicity	Lower immune activation, inflammation and toxicity
9	Application	Acute disease conditions; Where high doses are tolerable	Chronic, life threatening diseases or disorders; Where low doses are desirable

siRNA

siRNA is formed in the cell from shRNA or from long synthetic dsRNA by the Dicer enzyme, and is later separated into two short strands, one of which binds to the target mRNA and cleaves it, preventing the unwanted protein from being made. The first publication in 1998 by Nobel Prize winners Fire and Mello described RNA interference in worms induced by synthetic long double stranded RNA molecules. Synthetic siRNA was first described by Zamore et al in 2000, and remains the most widely used RNAi modality for gene silencing. In the siRNA approach, specific siRNAs are synthesized in the laboratory to silence specific proteins in target cells which are implicated in disease.

Because siRNA is composed of RNA and is inherently fragile, it is difficult to get into the target cell’s cytoplasm and, once there, is quickly degraded. This means that relatively high doses (in the nanomole range) are needed to achieve the desired level of gene silencing, and that treatment must be ongoing.
shRNA

shRNA is short hairpin RNA, double stranded RNA (dsRNA) which is created in the cell from a DNA construct encoding a sequence of single stranded RNA and its complement, separated by a stuffer fragment, allowing the RNA molecule to fold back on itself, creating a dsRNA molecule with a hairpin loop. (What is ddRNAi). In its synthetic form, shRNA suffers from the same constraints as outlined above for siRNA. However, when it is produced inside the cell from a DNA construct, it has the positive characteristics of siRNA, yet is produced continuously by the target cell’s own machinery.

The target cell can be directed to produce shRNA by specific DNA sequences introduced to the cell via a small gene cassette which travels to the nucleus. Here the introduced DNA either becomes part of the cell’s own DNA or persists in the nucleus, and instructs the cell to produce the specific shRNA, which is then processed by Dicer to siRNA and continues along the RNAi pathway via RISC to silence the gene.

Unlike siRNA, this DNA-directed shRNA mechanism requires a minute dose of DNA (in the range of 5 copies) yet the effect is long-lasting from a single dose. This is the RNAi modality for which Benitec Biopharma holds over 50 global patents.
^mce-anchor1 Advanced Drug Delivery Reviews 2009
siRNA vs. shRNA: Similarities and differences.
Rao DD, Vorhies JS, Senzer N, Nemunaitis J.
Advanced Drug Delivery Reviews 2009; 61: 746–759.

^mce-anchor2 Sci Trans Med 2010
RNA-based gene therapy for HIV with lentiviral vector-modified CD34+ cells in patients undergoing transplantation for AIDS-related lymphoma.
DiGiusto DL Krishnan A, Li L, Li H, Li S, Rao A, Mi S, Yam P, Stinson S, Kalos M, Alvarnas J, Lacey SF, Yee J-K, Li M, Couture L, Hsu D, Forman SJ, Rossi JJ, Zaia JA.
Sci Trans Med 2010. June; 2 Issue








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