What is Gene Silencing?

In lay terms, gene silencing the regulation of a gene that prevents it from expressing itself in a cell. Basically, gene silencing can reduce the expression of a gene in a cell by at least 70%, greatly lessening the impact that gene will have on the cell.

It is similar to gene knockdown which is when a gene is completely erased from an organism’s genome, resulting in no expression what so ever.

Gene silencing is often used in research to study genes associated with various disorders such as cancer, neurodegenerative diseases, respiratory diseases and infectious diseases. Related to this, gene silencing is also being used in drug research to try and find treatments for these conditions.

Gene silencing has been around for some time, but recent advancements have drawn more attention because scientists are getting much closer to being able to develop effective and accurate treatments for diseases that have previously been tagged as difficult to treat.

Many diseases including age-related macular degeneration, diabetes, kidney disease, cancer, Lou Gehrig's and Parkinson's have been tagged as candidates for gene silencing therapy, creating a wave of on-going clinical trials.

How is gene silencing done?

To understand gene silencing, you need to understand how genes work in the body. Proteins are generated at the molecular level and they act as tiny machines inside of cells.

They make chemical reactions take place, communicate messages and give cells their structure, among other things.

Each different protein is made using instructions called a gene. Genes are made from DNA and they live in the nucleus of each cell.

Genes do not make proteins directly. Instead, they use a DNA sequence of the gene as a template to make a message molecule.

This messenger molecule is called messenger RNA or mRNA. It is the mRNA that tells the cell what building blocks to use to create the protein.

When a gene is not functioning properly, it instructs the body to produce a version of the protein in question which can ultimately damage the cell.

There is a two-step process for a gene to produce a corresponding protein.

First a copy of the information is encoded in a gene made in the form of the mRNA. This process is known as transcription.

Transcription happens in the nucleus of the cell where all of the cells genetic material is contained.

The mRNA then travels outside of the nucleus along with the genetic information it carries which is used to produce a specific protein. This process in known as translation.

The idea behind gene silencing is to intervene in the gene expression prior to translation taking place. By designing a molecule that can specifically identify and breakdown the mRNA instructions for making a certain protein, scientists have been able to decrease, or silence, levels of that protein.

Essentially, the information needed to produce the protein in a cell is being blocked or censored in the mRNA message. When this information cannot be carried into a cell, it silences the gene that is providing these instructions.

By being able to significantly lower the levels of a specific protein, it opens up many possibilities in scientific research and drug development, since proteins are critically involved in the proper function and structure of cells.

How are proteins made in cells?

Proteins are one of life’s four essential building blocks. The other three are carbohydrates, lipids, and nucleic acids (DNA and RNA). Proteins can be either big such as part of the fibers that make up your muscles, or they can be small and be part of the structural elements of a cell.

Enzymes are a form of proteins that are essential for all kinds of reactions in your body, from the digestion of food to the replication of DNA. Proteins can also be antibodies that are used to help your immune system fight on infections or hormones that send messages throughout your body.

So, when you eat dietary protein, you body recycles this material and uses it to build all of the protein in your body.

For your body to make proteins, your DNA must provide instructions for making RNA. RNA is short for ribonucleic acid which then provides instructions for making protein.

RNA is similar to DNA. Just like DNA, it is made up of nucleotide subunits that contain a sugar molecule, a phosphate group and a nitrogenous base. But RNA also has an additional chemical group that makes it more reactive and less stable than DNA.

DNA always is found in the nucleus of complex cells only, but RNA is found both inside and outside of the nucleus.

To make a protein, a cell must put together a chain of amino acids in the right order. It makes a copy of the relevant DNA instructions and caries it into the cytoplasm of a cell.

Here is where the cell decodes the instructions and makes copies of the protein. The copy is made in the form of an RNA molecule.

It enters the cell cytoplasm where it is decoded. The RNA molecule is now known as mRNA.

Once in the cytoplasm, the mRNA is grabbed by tiny protein-assembly machines called ribosomes. Each ribosome works its way along the mRNA, reading the code from 'start' to 'stop', selecting the correct amino acid building blocks and ejecting a growing protein.

It takes just 1/50th of a second for the ribosome to select and add each building block. At this rate, a cell can assemble a small protein like insulin in just a few seconds.

Types of gene silencing methods

There are several types of gene silencing methods that researchers are pursuing.

Antisense oligonucleotides (ASO)

Antisense oligonucleotides (ASOs) are short, synthetic, single-stranded oligodeoxynucleotides that can alter RNA and reduce, restore, or modify protein expression through several distinct mechanisms.

More than two decades ago, researchers discovered that antisense oligonucleotides (ASOs) could influence RNA processing and modulate protein expression. Since that time, research has been hampered by a number of things such as insufficient biological activity, off-target toxic effects and by inadequate target engagement.

To address these issues, scientists have continued to introduce chemical modifications of ASOs to move toward a better solution. This steady progress has resulted in recent years of approvals of ASOs for the treatment of spinal muscular atrophy and Duchenne muscular dystrophy, representing landmarks in a field where disease-modifying therapies were virtually non-existent.

This technology holds the potential to change therapeutic treatments for many neurological and non-neurological conditions going forward.

RNAi-based therapies

RNA interference (RNAi) is a highly specific approach to gene silencing and has tremendous potential in the treatment of conditions such as cardiovascular diseases, viral infections, and cancer.

The RNA interference (RNAi) pathway regulates mRNA stability and translation in nearly all human cells. Small double-stranded RNA molecules can trigger RNAi silencing of specific genes, but their therapeutic use has faced a lot of issues involving safety and potency.

RNAi therapies are widely used in preclinical models, but the clinical application of RNAi remains a challenge because of the difficulty in achieving successful systemic delivery. Effective delivery systems are essential to enable the full therapeutic potential of RNAi.

An ideal nanocarrier needs to address chemically unstable features, extracellular and intracellular barriers, and innate immune stimulation, as well as offering "smart" targeted delivery. Over the past decade, great efforts have been undertaken to develop RNAi delivery systems that overcome these obstacles.

Progress is being made and was best evidenced in August 2018, with the US Food and Drug Administration approving patisiran, the first RNAi-based drug.

CRISPR/Cas9 system

The functions of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes are essential in adaptive immunity in select bacteria and archaea. These make it possible for organisms to respond and eliminate invading genetic materials.

They were first discovered in the 1980s in E. coli, but how they worked was not confirmed until 2007.

The simplicity of the CRISPR nuclease, with only three required components (Cas9 along with the crRNA and trRNA) makes this system amenable to adaptation for genome editing. To date, three different variants of the Cas9 nuclease have been adopted in genome-editing protocols.

Targeting efficiency is the percentage of desired mutation achieved and is one of the most important parameters to assess a genome-editing tool. In this case, the targeting efficiency of Cas9 compares favorably with more established methods running at 70% or more vs. 50% or less in other methods.

Following its initial demonstration in 2012, the CRISPR/Cas9 system has been widely adopted. It has already been successfully used to target important genes in many cell lines and organisms, including humans.

Due to the simplicity, high efficiency and versatility, the rapid progress in developing Cas9 into a set of tools for cell and molecular biology research has been remarkable.

Gene silencing and Huntington's disease (HD)

Huntington’s disease is also known as Huntington’s chorea and is an incurable inherited condition that causes motor, cognitive, and behavioral deficits as a result of the death of brain cells.

The disease starts out with subtle mood or mental problems and progresses to uncoordinated and jerky body movements. As the disease worsens movement becomes more difficult and a person is eventually unable to talk.

The disease is caused by a mutation in either of a person’s two copies of the Huntingtin gene.

The Huntingtin gene provides the genetic information for a protein that is also called "huntingtin".

Most people with HD, or those who develop it later, have one ‘normal’ gene and one with too many repeats of the three-letter sequence ‘CAG’ near the beginning. When this happens, it results in a ‘mutant’ protein that behaves differently from the normal protein, damaging cells and producing the symptoms of HD.

Gene silencing can be used to treat HD by targeting the mutant huntingtin protein.

In clinical trials involving mice, it was found that siRNA could reduce the normal and mutant huntingtin levels by 75%. When treated, mice developed improved motor control and lived longer.

Gene silencing and cancer

RNAi has been used to silence genes that are associated with several forms of cancer. Cancer comes in many forms, but there are a number of universal aberrations common to all cancers. One of these is the epigenetic silencing of tumor suppressor genes (TSGs).

The silencing of TSGs is believed to be an early, driving event in the development of cancer in humans.

As a result, efforts have been made to develop small molecules aimed at the restoration of TSGs to limit tumor cell proliferation and survival.

Recent evidence indicates that epigenetic changes might 'addict' cancer cells to altered pathways during the early stages of tumor development. Dependence on these pathways for cells to grow or survival allows them to acquire genetic mutations in the same pathways, providing the cell with characteristics that promote tumor progression.

Strategies to reverse epigenetic gene silencing could be useful in cancer prevention and therapy.

As an example, chemokine receptor chemokine receptor 4 (CXCR4), associated with the proliferation of breast cancer, was cleaved by siRNA molecules that reduced the number of divisions commonly observed by the cancer cells. Researchers have also used siRNAs to selectively regulate the expression of cancer-related genes.

Studies are also being increasingly utilized to study the potential use of siRNA molecules in cancer therapeutics. For instance, mice implanted with colon adenocarcinoma cells were found to survive longer when the cells were pretreated with siRNAs that targeted B-catenin in the cancer cells.

Gene-silencing drugs and FDA approval

After years and years of research, the first ever gene-silencing drug won Food and Drug Administration approval in 2018.

Capitalizing on a Nobel Prize-winning discovery, scientists were able to deliver on the medical payoff that takes place when RNA molecules can silence genes by interrupting the translation of DNA’s instructions into proteins.

The new drug treats hereditary transthyretin amyloidosis, or ATTR, which affects about 50,000 people worldwide. ATTR is a hereditary condition that causes misshapen proteins to build up in patients’ nerves, tissues and organs, causing loss of sensation, organ failure and even death.

The drug is called patisiran and used specially engineered pieces of RNA to silence a mutated gene when it is active in the liver. It is not a cure for ATTR, and people still have the genetic mutation, but the treatment was shown to prevent the disease from spreading.

Scientists are elated at the results because it signals the first of many possible gene silencing drugs that can be brought to the public. Many more drugs using the same approach are winding through clinical trials for diseases ranging from hemophilia to HIV.

Patisiran and other RNA interference-based therapies that are being developed use specially crafted snippets of synthetic RNA to artificially manipulate genes’ activity.

One of the attractive benefits of creating RNA interference drugs is the theoretical simplicity of it. But this has been offset by the reality that took researchers decades to figure out trying to determine how to deliver these drugs to the right place in the body to reduce harmful off-target side effects, and how to design synthetic RNA molecules that don’t degrade before they do their job.

Gene silencing therapy challenges and possible side effects

In the big picture, while a lot of progress has been made regarding gene silencing research, there is still a lot that is unknown.

These unknowns have uncovered certain challenges and side effects that need to be addressed as research and implementation moves forward.

Much of the challenges involve delivery and specificity. For example, in the case of neurodegenerative disorders, gene silencing molecules must be delivered to the brain.

But there is a blood-brain barrier that makes it difficult to delivery molecules to the brain. As a result, researchers must resort to injecting molecules or implant pumps that push these molecules directly into the brain.

Once inside the brain, the molecules must move inside of the targeted cells which is done through viral vectors. But this method of delivery can be a problem since it may create an immune response against the molecules.

The other issue of specificity means that both ASOs and siRNA molecules can potentially bind to the wrong mRNA molecule.

Also, in 2018, givosiran was introduced in the marketplace and met the main goal of reducing the yearly number of attacks in patients with acute intermittent porphyria (AIP) - a disease that affects the liver and causes debilitating attacks that render most disabled.

Givosiran uses RNA interference (RNAi), to target and silence specific genetic material, blocking the production of the deadly protein that causes the disease.

But a large portion of the patients being treated with givosiran experienced serious side effects, such as renal impairment and elevated liver enzymes, compared to those on placebo.

These two examples are part of a larger indication that there is a continued need for caution in current clinical trials using the technology, as it may have potentially harmful effects on subjects.

Researchers continue to look for more efficient methods to deliver and develop specific gene silencing therapeutics that are still safe and effective.

Advantages of gene silencing

Some of the advantages of gene silencing include:

• Cost effective
• Powerful tool for analyzing unknown genes in sequenced genomes.
• Useful approach in future gene therapy.
• With Si RNA, the researcher can simultaneously perform experiments in any cell type of interest
• Blocking expression of unwanted genes and undesirable substances.
• Inducing viral resistance
• Oligonucleotides can be manufactured quickly, some within one week; the sequence of the mRNA is all that is needed
• Downregulation of gene expression simplifies "knockout" analysis.
• Easier than use of antisense oligonucleotides. Si RNA more effective and sensitive at lower concentration.

Disadvantages of gene silencing

Some of the disadvantages of gene silencing include:

• High pressure injection” and electroporation can cause significant injection damage to the integrity of the normal tissues and organs and thus preclude the utilization in a clinical set-up.
• Liposomes/cationic encapsulated Si RNA may also be toxic to the host and may cause severe host immune responses.
• Other emerging strategies includes chemical modification of Si RNA molecules and encapsulated with different molecules are still in their infancy and need to be thoroughly investigated before used in therapeutic applications.
• Can be hard to target specific molecules at times.
• Delivery to certain areas of the body can be challenging

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