DNA-directed RNA interference

Unlike small interfering RNAs (siRNA) that turn over within a cell and silence genes transiently, DNA constructs are continually transcribed, replenishing the cellular supply of siRNAs. This allows for the long-term silencing of targeted genes, which has the potential for ongoing clinical benefit with reduced medical intervention.

File:ddRNAi diagram.jpg (or reagent) tailored to target specific cells. Inside the cell, the DNA is transported to the nucleus where transcription machinery continually manufactures the encoded RNAs. The shRNA molecules are then processed by endogenous systems before entering the RNAi pathway and silencing the desired genes.]]

Figure 1 illustrates the most common type of ddRNAi DNA construct designed to express a shRNA. This figure consists of a promoter sequence driving the expression of sense and antisense sequences separated by a loop sequence, followed by a transcriptional terminator. The antisense sequence processed from the shRNA can bind to the target RNA and specify its degradation. shRNA constructs typically encode sense and antisense sequences of 20–30 nucleotides. Flexibility in construct design is possible; for example, the positions of sense and antisense sequences can be reversed, and other modifications and additions can alter intracellular shRNA processing.{{Cite journal | last1 = Gu | first1 = S. | last2 = Jin | first2 = L. | last3 = Zhang | first3 = Y. | last4 = Huang | first4 = Y. | last5 = Zhang | first5 = F. | last6 = Valdmanis | first6 = P. N. | last7 = Kay | first7 = M. A. | doi = 10.1016/j.cell.2012.09.042 | title = The Loop Position of shRNAs and Pre-miRNAs is Critical for the Accuracy of Dicer Processing In Vivo | journal = Cell | volume = 151 | issue = 4 | pages = 900–911 | year = 2012 | pmid = 23141545 | pmc =3499986 }} Moreover, a variety of promoter loop and terminator sequences can be used.

File:ddRNAi constructs jpg.jpg

A variant of this is the multi-cassette (Figure 2b). Designed to express two or more shRNAs, it can target multiple sequences for degradation simultaneously, which can be beneficial in circumstances such as when targeting viruses. Natural sequence variations can render a single shRNA-target site unrecognizable, preventing RNA degradation. Multi-cassette constructs that target multiple sites within the same viral RNA circumvent this issue.

Delivery of ddRNAi DNA constructs is a major challenge for RNAi-based therapy. There are a number of clinically-approved gene therapy vectors developed for therapeutic use. Two broad strategies to facilitate the delivery of DNA constructs to the desired cells are available: these use either viral vectors or one of several classes of transfection reagents.

In vivo delivery of ddRNAi constructs has been demonstrated using a range of vectors and reagents with different routes of administration (ROA). ddRNAi constructs have also been successfully delivered into host cells ex vivo, and then transplanted back into the host.{{Cite journal

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For example, in a phase I clinical trial at the City of Hope National Medical Center, California, four HIV-positive patients with non-Hodgkin's lymphoma were successfully treated with autologous hematopoietic progenitor cells pre-transduced ex vivo with ddRNAi constructs using lentiviral vectors. This construct was designed to express three therapeutic RNAs, one of which was a shRNA, thereby combating HIV replication in three different ways:

• shRNA, silencing the tat and rev genes of the HIV genome;
• CCR5 ribozyme, inhibiting viral cell entry;
• TAR decoy RNA, inhibiting initiation of viral transcription.

Ongoing expression of the shRNA has been confirmed in T cells, monocytes, and B cells more than one year after transplantation.

Nervana is an investigational V construct that knocks down the expression of protein kinase C gamma (PKCγ) known to be associated with neuropathic pain and morphine tolerance.

Two conserved PKCγ sequences across all key model species and humans have been identified, and both single and double DNA cassettes are designed. In vitro, the expression of PKCγ was silenced by 80%. When similar ddRNAi constructs were delivered intrathecally using a lentiviral vector, pain relief in a neuropathic-rat model was demonstrated.

The development of resistance to chemotherapies such as paclitaxel and cisplatin in non-small-cell lung cancer (NSCLC) is strongly associated with overexpression of beta III tubulin. Investigations by the Children's Cancer Institute Australia (University of New South Wales, Lowy Cancer Research Centre) demonstrated that beta III-tubulin knockdown by ddRNAi delayed tumor growth and increased chemosensitivity in mouse models.

Tributarna is a triple DNA cassette expressing three shRNA molecules each of which separately targets beta III tubulin and strongly inhibits its expression. Studies in an orthotopic-mouse model, where the construct is delivered by a modified polyethylenimine vector, jetPEI, that targets lung tissue are in progress.{{Citation needed|date=October 2024}}

The hepatitis B virus (HBV) genome encodes its own DNA polymerase for replication. Biomics Biotechnologies has evaluated around 5000 siRNA sequences of this gene for effective knockdown; five sequences were chosen for further investigation and were shown to have silencing activity when converted into shRNA expression cassettes. A multi-cassette construct, Hepbarna, is under preclinical development for delivery by an adeno-associated virus 8 (AAV-8) liver-targeting vector.{{Citation needed|date=October 2024}}

Classified as an orphan disease, there is currently no therapy for oculopharyngeal muscular dystrophy (OPMD), as it is caused by a mutation in the poly(A) binding protein nuclear 1 (PABPN1) gene. Silencing the mutant gene using ddRNAi offers a potential therapeutic approach.{{Cite journal

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Besides the ex vivo approach discussed above, the Center for Infection and Immunity Amsterdam (CINIMA) of the University of Amsterdam, Netherlands, is extensively researching the therapeutic potential of multi-cassette DNA constructs for HIV.{{Cite journal

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As with all gene therapies, a number of safety and toxicity issues must be evaluated during the development of ddRNAi therapeutic techniques.

Some gene therapy vectors integrate into the host genome, thereby acting as insertional mutagens. This was a particular issue with early retroviral vectors, where insertions adjacent to oncogenes resulted in the development of lymphoid tumors.{{Cite journal

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Adeno-associated virus (AAV) vectors are considered low-risk for host-genome integration, as AAV infection has not been associated with the induction of cancers in humans despite widespread prevalence across the general population. Moreover, extensive clinical use of AAV vectors has provided no evidence of carcinogenicity. While lentiviral vectors do integrate into the genome, they do not appear to show a propensity to activate oncogene expression.{{Cite journal

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An immunological response to an adenoviral vector resulted in the death of a patient in an early human trial. Careful monitoring of potential toxicities in preclinical testing and analysis of any pre-existing antibodies to gene therapy vectors in patients minimizes such risks.{{Citation needed|date=October 2024}}

siRNAs have been shown to activate immune responses through interaction with toll-like receptors (TLRs), leading to interferon responses. These TLRs reside on the cell's outer surface, so ddRNAi constructs, which are delivered directly into the intracellular space, are not expected to induce such a response.{{Cite journal

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High-level expression of shRNAs has been shown to be toxic. Strategies to minimize levels of shRNA expression or promote precise processing of shRNAs{{Cite journal

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The unintended silencing of genes that share sequence homology with expressed shRNAs could theoretically occur.{{Cite journal

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}} Careful selection of shRNA sequences and thorough preclinical testing of constructs can mitigate this risk.

• {{Cite book | last1 = Rice | first1 = R. R. | last2 = Muirhead | first2 = A. N. | last3 = Harrison | first3 = B. T. | last4 = Kassianos | first4 = A. J. | last5 = Sedlak | first5 = P. L. | last6 = Maugeri | first6 = N. J. | last7 = Goss | first7 = P. J. | last8 = Davey | first8 = J. R. | last9 = James | first9 = D. E. | last10 = Graham | first10 = M. W. | chapter = Simple, Robust Strategies for Generating DNA-Directed RNA Interference Constructs | title = RNA Interference | series = Methods in Enzymology | doi = 10.1016/S0076-6879(04)92024-1 | volume = 392 | pages = 405–419 | year = 2005 | isbn = 9780121827977 | pmid = 15644195 }}
• {{Cite book | last1 = Liu | first1 = Y. P. | last2 = Westerink | first2 = J. T. | last3 = Brake | first3 = O. | last4 = Berkhout | first4 = B. | doi = 10.1007/978-1-61779-037-9_18 | chapter = RNAi-Inducing Lentiviral Vectors for Anti-HIV-1 Gene Therapy | title = Antiviral RNAi | series = Methods in Molecular Biology | volume = 721 | pages = 293–311 | year = 2011 | isbn = 978-1-61779-036-2 | pmid = 21431693 }}

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