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Genome editing (also called gene editing) is a group of technologies that give scientists the ability to change an organism’s DNA. These technologies allow genetic material to be added, removed, or altered at particular locations in the genome. Several approaches to genome editing have been developed. A well-known one is called CRISPR-Cas9, which is short for clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9. The CRISPR-Cas9 system has generated a lot of excitement in the scientific community because it is faster, cheaper, more accurate, and more efficient than other genome editing methods.

CRISPR-Cas9 was adapted from a naturally occurring genome editing system that bacteria use as an immune defense. When infected with viruses, bacteria capture small pieces of the viruses’ DNA and insert them into their own DNA in a particular pattern to create segments known as CRISPR arrays. The CRISPR arrays allow the bacteria to “remember” the viruses (or closely related ones). If the viruses attack again, the bacteria produce RNA segments from the CRISPR arrays that recognize and attach to specific regions of the viruses’ DNA. The bacteria then use Cas9 or a similar enzyme to cut the DNA apart, which disables the virus.

Researchers adapted this immune defense system to edit DNA. They create a small piece of RNA with a short “guide” sequence that attaches (binds) to a specific target sequence in a cell’s DNA, much like the RNA segments bacteria produce from the CRISPR array. This guide RNA also attaches to the Cas9 enzyme. When introduced into cells, the guide RNA recognizes the intended DNA sequence, and the Cas9 enzyme cuts the DNA at the targeted location, mirroring the process in bacteria. Although Cas9 is the enzyme that is used most often, other enzymes (for example Cpf1) can also be used. Once the DNA is cut, researchers use the cell’s own DNA repair machinery to add or delete pieces of genetic material, or to make changes to the DNA by replacing an existing segment with a customized DNA sequence.

Genome editing is of great interest in the prevention and treatment of human diseases. Currently, genome editing is used in cells and animal models in research labs to understand diseases. Scientists are still working to determine whether this approach is safe and effective for use in people. It is being explored in research and clinical trials for a wide variety of diseases, including single-gene disorders such as cystic fibrosis, hemophilia, and sickle cell disease. It also holds promise for the treatment and prevention of more complex diseases, such as cancer, heart disease, mental illness, and human immunodeficiency virus (HIV) infection.

Ethical concerns arise when genome editing, using technologies such as CRISPR-Cas9, is used to alter human genomes. Most of the changes introduced with genome editing are limited to somatic cells, which are cells other than egg and sperm cells (germline cells). These changes are isolated to only certain tissues and are not passed from one generation to the next. However, changes made to genes in egg or sperm cells or to the genes of an embryo could be passed to future generations. Germline cell and embryo genome editing bring up a number of ethical challenges, including whether it would be permissible to use this technology to enhance normal human traits (such as height or intelligence). Based on concerns about ethics and safety, germline cell and embryo genome editing are currently illegal in the United States and many other countries.

Several treatments have been developed that involve genetic material but are typically not considered gene therapy. Some of these methods alter DNA for a slightly different use than gene therapy. Others do not alter genes themselves, but they change whether or how a gene’s instructions are carried out to make proteins.

Cell-based gene therapy

CAR T cell therapy (or chimeric antigen receptor T cell therapy) is an example of cell-based gene therapy. This type of treatment combines the technologies of gene therapy and cell therapy. Cell therapy introduces cells to the body that have a particular function to help treat a disease. In cell-based gene therapy, the cells have been genetically altered to give them the special function. CAR T cell therapy introduces a gene to a person’s T cells, which are a type of immune cell. This gene provides instructions for making a protein, called the chimeric antigen receptor (CAR), that attaches to cancer cells. The modified immune cells can specifically attack cancer cells.

RNA therapy

Several techniques, called RNA therapies, use pieces of RNA, which is a type of genetic material similar to DNA, to help treat a disorder. In many of these techniques, the pieces of RNA interact with a molecule called messenger RNA (or mRNA for short). In cells, mRNA uses the information in genes to create a blueprint for making proteins. By interacting with mRNA, these therapies influence how much protein is produced from a gene, which can compensate for the effects of a genetic alteration. Examples of these RNA therapies include antisense oligonucleotide (ASO), small interfering RNA (siRNA), and microRNA (miRNA) therapies. An RNA therapy called RNA aptamer therapy introduces small pieces of RNA that attach directly to proteins to alter their function.

Epigenetic therapy

Another gene-related therapy, called epigenetic therapy, affects epigenetic changes in cells. Epigenetic changes are specific modifications (often called “tags”) attached to DNA that control whether genes are turned on or off. Abnormal patterns of epigenetic modifications alter gene activity and, subsequently, protein production. Epigenetic therapies are used to correct epigenetic errors that underlie genetic disorders.

Scientific journal articles for further reading

Kim YK. RNA Therapy: Current Status and Future Potential. Chonnam Med J. 2020 May;56(2):87-93. doi: 10.4068/cmj.2020.56.2.87. Epub 2020 May 25. PubMed: 32509554. Free full-text article from PubMed Central: PMC7250668.

Lu Y, Chan YT, Tan HY, Li S, Wang N, Feng Y. Epigenetic regulation in human cancer: the potential role of epi-drug in cancer therapy. Mol Cancer. 2020 Apr 27;19(1):79. doi: 10.1186/s12943-020-01197-3. 32340605. Free full-text article from PubMed Central: PMC7184703.

Use of Genetic Material

As we describe the different approaches of gene and cell therapy we often say “genetic material” is used or delivered to cells. Genetic material is a broad term and there are different types that may be used.

Genetic Material most commonly refers to DNA or RNA. These are strings of molecules with the information to instruct cells to produce proteins. Proteins play an important role in how our body functions. 

DNA stores the genetic information so the cell can continue building proteins. It is permanent and stored in the nucleus in a form called chromosomes. DNA is made up of two strands and is much larger than RNA. Genes are specific sections of DNA that encode sequences (I.e. a set of instructions) for making proteins. Other sections of DNA can control when, where, and how much of the protein is made. 

RNA is a copy of the genetic information contained within DNA. While DNA is the permanent storage format of genetic material, RNA molecules help turn those instructions into proteins. After RNA is made within the nucleus of a cell, most RNA moves to the cytoplasm, which is the fluid space between the nucleus and cell membrane. There are many different types of RNA. Some RNA carries the instructions for making proteins, while other types of RNA can prevent proteins from being made. RNA molecules are only active for a limited period of time in the cell.  Keep reading to learn more about the types of RNA used in RNA Therapies.

Breaking Down the Approaches

Gene Therapy is the use of genetic material to treat or prevent disease. Learn more about Gene therapy Basics or Vectors 101. Three common effects of gene therapies in cells are: 

• Gene addition ​adds in a working gene that has the instructions for the cell to make more of the specific protein needed. Vectors, which are often viruses, are used to deliver the working gene to the cell’s nucleus, where the DNA is stored. This gene will now live in the nucleus which gives a greater chance of being permanent and is only given one time. Sometimes the therapy is designed for the new gene to insert itself into the main DNA storage while other times it will stay next to the main DNA storage, like an extra set of instructions.
• Gene silencing is where the delivered genetic material prevents or inhibits the activity of a gene that is already present in a cell. Gene silencing often decreases the amount of a specific protein being made. 
• Gene Editing corrects pieces of DNA by changing or deleting the information within the affected individual’s gene. Genetic material is sent to directly edit or change pieces of DNA already located within a cell to correct the protein being made by that DNA. Gene editing uses technology that is highly precise to make these types of changes. Learn more about Gene Editing.

Please note, gene therapy is a rapidly evolving field that changes in response to new scientific discoveries and from what is learned about treatments being tested in the clinic and laboratory.  Below we highlight different types of gene therapies and strategies that are currently being tested and may be approved for use in humans.  As DNA and RNA work together, there may be overlap in the way each is applied.

DNA therapy is the use of DNA that codes for the production of a specific RNA or protein to treat a disorder.  To have a therapeutic effect, the DNA must be delivered to the nucleus of a cell, where it can then be used by the cell to affect protein expression.  This gene will now live in the nucleus which gives a greater chance of being permanent and is typically only given one time. 

• DNA plasmids are large, double-stranded circular DNA molecules that code for a therapeutic protein.  DNA plasmids can be used for gene addition, vaccination, and cell therapy.  A challenge for DNA plasmids is getting them into the cell and then into the nucleus for therapeutic benefit.  Clinical trials are ongoing to test the safety and benefit of DNA plasmid therapeutics.
• Viral vectors are modified viruses used as vehicles to deliver therapeutic genetic material or specific DNA sequences into a cell.  In a viral vector, the viral genes are removed and replaced by a therapeutic DNA sequence encodes genes, RNAs or other substances and packaged inside the shell of a virus.  The viral vector is then delivered directly to the body (in-vivo therapy) or to cells (ex-vivo therapy; see Gene-modified cell therapy below) to deliver the therapeutic genetic material to the nucleus of the cell. ​In-vivo viral vector therapies are frequently limited to a one-time delivery due to the innate immune response to the virus that usually prevents re-dosing. Learn more about innate immunity in Vectors 101. There are multiple FDA approved viral vector DNA therapies.

RNA therapy is the use of shorter sequences of genetic material in RNA format to treat or prevent a disease. There are many different types of RNA therapy because there are so many different types of RNA sequences that can affect cell functions. These types of therapies are delivered using viral vectors, or other non-viral vehicles such as lipid nanoparticles. They often need repeat dosing to maintain a therapeutic (good) effect since the DNA is not being altered or supplemented.  Let’s explore the different types of RNA and corresponding therapies: 

• Ribosomal RNA (rRNA) is theRNA that helps form ribosomes, which are the molecular machines used in building proteins. 
• Messenger RNA (mRNA) is a middle message that can move through different parts of the cell to provide instructions to make proteins. It is a single stand that carries the information, initially stored within DNA, out of the nucleus to the cytoplasm of a cell where proteins are made.

  • mRNA therapy is designed to produce more of a specific protein when the gene for that protein is missing, not working the way it should, or is beneficial for our bodies to create. Lipid nanoparticles may be used as containers to deliver therapeutic mRNA into cells because its structure protects the contents from being degraded after being injected into the body. Learn more about mRNA in vaccines.

• microRNA (miRNA) is a small form of single-strand RNA that typically targets multiple mRNAs to regulate expression of several different genes at the same time.  

  • miRNA therapies can be in the form of synthetic, double-stranded miRNAs (also called miRNA mimics), recombinant expression vectors that carry miRNA encoding sequences (naturally occurring or artificial), and oligonucleotide-based inhibitors (anti-miRs).  Testing of miRNA therapies are ongoing in clinical trials.

• Small interfering RNA (siRNA) are double stranded RNA molecules that usually target a specific mRNA to prevent production of unwanted proteins. 

  • siRNA therapies are designed outside of the body to target expression of a specific gene.  There are FDA approved siRNA therapies.

• Transfer RNA (tRNA) carry the building blocks of proteins, called amino acids, to the ribosome to help make a protein based off the mRNA instructions. 

  • Suppressor tRNA therapies are designed to override incorrect mRNA instructions that may cause disease by stopping protein production too early.  Binding of suppressor tRNA to the incorrectly made mRNA allows the correct, full protein to be made by the ribosome.  Suppressor tRNA therapies are still in the preclinical phase.

• Antisense oligonucleotides (ASO’s) are synthetic (fake), single-stranded chains of molecules that target mRNA from a specific gene.

  • ASO therapies act inside a cell to alter how proteins are made. ASOs can silence a gene so a protein is not made. It can also alter how mRNA is made to then change how a protein is made. These stay in the cell for a limited duration and may need to be given repeatedly to maintain a therapeutic effect. There are multiple FDA approved ASO therapies.

• RNA aptamers are short pieces of RNA that bind to a specific protein to control their functions (for example, blocking or activating). 

  • Aptamer therapies are designed outside of the body. Unlike other RNA based therapeutics that must be brought inside a cell to be beneficial, RNA aptamers can tightly bind to proteins on the outside of the cell to provide unique therapeutic advantages. There are FDA approved RNA apatmers.