DNA-directed RNA interference

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DNA-directed RNA interference (ddRNAi) is a gene-silencing technique that utilizes DNA constructs to activate a cell's endogenous RNA interference (RNAi) pathways. DNA constructs are designed to express self-complementary double-stranded RNAs, typically short-hairpin RNAs (shRNA), that bring about the silencing of a target gene or genes once processed. [1] Any RNA, including endogenous messenger RNA (mRNAs) or viral RNAs, can be silenced by designing constructs to express double-stranded RNA complementary to the desired mRNA target.

Contents

This mechanism has been recently demonstrated to work therapeutically to silence disease-causing genes across a range of disease models, including viral diseases such as HIV, [2] hepatitis B [3] or hepatitis C, [4] and diseases associated with altered expression of endogenous genes such as drug-resistant lung cancer, [5] neuropathic pain, [6] advanced cancer, [7] and retinitis pigmentosa. [8]

ddRNAi mechanism

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. [2] [4]

Figure 1: A ddRNAi construct encoding a shRNA is packaged into a delivery vector (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. DdRNAi diagram.jpg
Figure 1: A ddRNAi construct encoding a shRNA is packaged into a delivery vector (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.

Organization of ddRNAi constructs

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. [9] Moreover, a variety of promoter loop and terminator sequences can be used. [4]

Figure 2: ddRNAi DNA constructs DdRNAi constructs jpg.jpg
Figure 2: ddRNAi DNA constructs

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. [4]

Delivery

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). [3] [4] [6] [8] ddRNAi constructs have also been successfully delivered into host cells ex vivo, and then transplanted back into the host. [2] [7] [10]

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: [2]

Ongoing expression of the shRNA has been confirmed in T cells, monocytes, and B cells more than one year after transplantation. [2]

Therapeutic applications

Neuropathic pain

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. [6]

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. [6]

Drug-resistant non-small-cell lung cancer

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. [5]

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 ]

Hepatitis B viral infection

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 ]

Oculopharyngeal muscular dystrophy

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. [11]

HIV/AIDS

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. [12]

Concerns

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

Oncogene activation by viral insertion

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. [13]

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. [14]

Immune response to gene therapy vectors

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 ]

Innate immune response

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. [15]

Toxic effects due to over-expression of shRNAs

High-level expression of shRNAs has been shown to be toxic. Strategies to minimize levels of shRNA expression [4] or promote precise processing of shRNAs [16] can mitigate this issue.

Off-target effects

The unintended silencing of genes that share sequence homology with expressed shRNAs could theoretically occur. [17] Careful selection of shRNA sequences and thorough preclinical testing of constructs can mitigate this risk.

Further reading

Related Research Articles

<span class="mw-page-title-main">Gene therapy</span> Medical technology

Gene therapy is a medical technology that aims to produce a therapeutic effect through the manipulation of gene expression or through altering the biological properties of living cells.

Gene silencing is the regulation of gene expression in a cell to prevent the expression of a certain gene. Gene silencing can occur during either transcription or translation and is often used in research. In particular, methods used to silence genes are being increasingly used to produce therapeutics to combat cancer and other diseases, such as infectious diseases and neurodegenerative disorders.

Gene knockdown is an experimental technique by which the expression of one or more of an organism's genes is reduced. The reduction can occur either through genetic modification or by treatment with a reagent such as a short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript.

<span class="mw-page-title-main">Small interfering RNA</span> Biomolecule

Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded non-coding RNA molecules, typically 20–24 base pairs in length, similar to microRNA (miRNA), and operating within the RNA interference (RNAi) pathway. It interferes with the expression of specific genes with complementary nucleotide sequences by degrading messenger RNA (mRNA) after transcription, preventing translation. It was discovered in 1998 by Andrew Fire at the Carnegie Institution for Science in Washington, D.C. and Craig Mello at the University of Massachusetts in Worcester.

<span class="mw-page-title-main">Dicer</span> Enzyme that cleaves double-stranded RNA (dsRNA) into short dsRNA fragments

Dicer, also known as endoribonuclease Dicer or helicase with RNase motif, is an enzyme that in humans is encoded by the DICER1 gene. Being part of the RNase III family, Dicer cleaves double-stranded RNA (dsRNA) and pre-microRNA (pre-miRNA) into short double-stranded RNA fragments called small interfering RNA and microRNA, respectively. These fragments are approximately 20–25 base pairs long with a two-base overhang on the 3′-end. Dicer facilitates the activation of the RNA-induced silencing complex (RISC), which is essential for RNA interference. RISC has a catalytic component Argonaute, which is an endonuclease capable of degrading messenger RNA (mRNA).

The RNA-induced silencing complex, or RISC, is a multiprotein complex, specifically a ribonucleoprotein, which functions in gene silencing via a variety of pathways at the transcriptional and translational levels. Using single-stranded RNA (ssRNA) fragments, such as microRNA (miRNA), or double-stranded small interfering RNA (siRNA), the complex functions as a key tool in gene regulation. The single strand of RNA acts as a template for RISC to recognize complementary messenger RNA (mRNA) transcript. Once found, one of the proteins in RISC, Argonaute, activates and cleaves the mRNA. This process is called RNA interference (RNAi) and it is found in many eukaryotes; it is a key process in defense against viral infections, as it is triggered by the presence of double-stranded RNA (dsRNA).

Lentivirus is a genus of retroviruses that cause chronic and deadly diseases characterized by long incubation periods, in humans and other mammalian species. The genus includes the human immunodeficiency virus (HIV), which causes AIDS. Lentiviruses are distributed worldwide, and are known to be hosted in apes, cows, goats, horses, cats, and sheep as well as several other mammals.

<span class="mw-page-title-main">Short hairpin RNA</span> Type of RNA

A short hairpin RNA or small hairpin RNA is an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi). Expression of shRNA in cells is typically accomplished by delivery of plasmids or through viral or bacterial vectors. shRNA is an advantageous mediator of RNAi in that it has a relatively low rate of degradation and turnover. However, it requires use of an expression vector, which has the potential to cause side effects in medicinal applications.

<span class="mw-page-title-main">Viral vector</span> Biotechnology to deliver genetic material into a cell

Viral vectors are modified viruses designed to deliver genetic material into cells. This process can be performed inside an organism or in cell culture. Viral vectors have widespread applications in basic research, agriculture, and medicine.

Therapeutic gene modulation refers to the practice of altering the expression of a gene at one of various stages, with a view to alleviate some form of ailment. It differs from gene therapy in that gene modulation seeks to alter the expression of an endogenous gene whereas gene therapy concerns the introduction of a gene whose product aids the recipient directly.

<span class="mw-page-title-main">Gene delivery</span> Introduction of foreign genetic material into host cells

Gene delivery is the process of introducing foreign genetic material, such as DNA or RNA, into host cells. Gene delivery must reach the genome of the host cell to induce gene expression. Successful gene delivery requires the foreign gene delivery to remain stable within the host cell and can either integrate into the genome or replicate independently of it. This requires foreign DNA to be synthesized as part of a vector, which is designed to enter the desired host cell and deliver the transgene to that cell's genome. Vectors utilized as the method for gene delivery can be divided into two categories, recombinant viruses and synthetic vectors.

RNA silencing or RNA interference refers to a family of gene silencing effects by which gene expression is negatively regulated by non-coding RNAs such as microRNAs. RNA silencing may also be defined as sequence-specific regulation of gene expression triggered by double-stranded RNA (dsRNA). RNA silencing mechanisms are conserved among most eukaryotes. The most common and well-studied example is RNA interference (RNAi), in which endogenously expressed microRNA (miRNA) or exogenously derived small interfering RNA (siRNA) induces the degradation of complementary messenger RNA. Other classes of small RNA have been identified, including piwi-interacting RNA (piRNA) and its subspecies repeat associated small interfering RNA (rasiRNA).

RNA activation (RNAa) is a small RNA-guided and Argonaute (Ago)-dependent gene regulation phenomenon in which promoter-targeted short double-stranded RNAs (dsRNAs) induce target gene expression at the transcriptional/epigenetic level. RNAa was first reported in a 2006 PNAS paper by Li et al. who also coined the term "RNAa" as a contrast to RNA interference (RNAi) to describe such gene activation phenomenon. dsRNAs that trigger RNAa have been termed small activating RNA (saRNA). Since the initial discovery of RNAa in human cells, many other groups have made similar observations in different mammalian species including human, non-human primates, rat and mice, plant and C. elegans, suggesting that RNAa is an evolutionarily conserved mechanism of gene regulation.

Gene therapy using lentiviral vectors was being explored in early stage trials as of 2009.

<span class="mw-page-title-main">RNA interference</span> Biological process of gene regulation

RNA interference (RNAi) is a biological process in which RNA molecules are involved in sequence-specific suppression of gene expression by double-stranded RNA, through translational or transcriptional repression. Historically, RNAi was known by other names, including co-suppression, post-transcriptional gene silencing (PTGS), and quelling. The detailed study of each of these seemingly different processes elucidated that the identity of these phenomena were all actually RNAi. Andrew Fire and Craig C. Mello shared the 2006 Nobel Prize in Physiology or Medicine for their work on RNAi in the nematode worm Caenorhabditis elegans, which they published in 1998. Since the discovery of RNAi and its regulatory potentials, it has become evident that RNAi has immense potential in suppression of desired genes. RNAi is now known as precise, efficient, stable and better than antisense therapy for gene suppression. Antisense RNA produced intracellularly by an expression vector may be developed and find utility as novel therapeutic agents.

<span class="mw-page-title-main">Cas9</span> Microbial protein found in Streptococcus pyogenes M1 GAS

Cas9 is a 160 kilodalton protein which plays a vital role in the immunological defense of certain bacteria against DNA viruses and plasmids, and is heavily utilized in genetic engineering applications. Its main function is to cut DNA and thereby alter a cell's genome. The CRISPR-Cas9 genome editing technique was a significant contributor to the Nobel Prize in Chemistry in 2020 being awarded to Emmanuelle Charpentier and Jennifer Doudna.

<span class="mw-page-title-main">Lentiviral vector in gene therapy</span> Method of gene modification

Lentiviral vectors in gene therapy is a method by which genes can be inserted, modified, or deleted in organisms using lentiviruses.

Since antiretroviral therapy requires a lifelong treatment regimen, research to find more permanent cures for HIV infection is currently underway. It is possible to synthesize zinc finger nucleotides with zinc finger components that selectively bind to specific portions of DNA. Conceptually, targeting and editing could focus on host cellular co-receptors for HIV or on proviral HIV DNA.

<span class="mw-page-title-main">Genome-wide CRISPR-Cas9 knockout screens</span> Research tool in genomics

Genome-wide CRISPR-Cas9 knockout screens aim to elucidate the relationship between genotype and phenotype by ablating gene expression on a genome-wide scale and studying the resulting phenotypic alterations. The approach utilises the CRISPR-Cas9 gene editing system, coupled with libraries of single guide RNAs (sgRNAs), which are designed to target every gene in the genome. Over recent years, the genome-wide CRISPR screen has emerged as a powerful tool for performing large-scale loss-of-function screens, with low noise, high knockout efficiency and minimal off-target effects.

<span class="mw-page-title-main">Intracellular delivery</span> Scientific research area

Intracellular delivery is the process of introducing external materials into living cells. Materials that are delivered into cells include nucleic acids, proteins, peptides, impermeable small molecules, synthetic nanomaterials, organelles, and micron-scale tracers, devices and objects. Such molecules and materials can be used to investigate cellular behavior, engineer cell operations or correct a pathological function.

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