How does gene therapy work?

Vectors

In gene therapy, genetic material from outside the body needs to get inside the nucleus. Currently, the only way this can happen is through the use of viral or non-viral vectors. You can learn more about vectors in this part of genehome.

What is a vector?

Gene therapy products work by introducing genetic material into the nucleus of the cell. In order to introduce the genetic material, scientists need a delivery system that can transport the gene, nuclease, or short hairpin RNA (shRNA) to the nucleus of a human cell. The vehicle that carries this genetic material is known as a vector. Like a microscopic delivery truck, the vector can transport and deliver genetic material to target cells or locations in the person’s genome.

There are two types of vectors, viral and non-viral. Viral vectors are currently a delivery vehicle used in FDA-approved gene therapies. Non-viral techniques are currently being studied as a safe and effective way to deliver genetic material to cells for therapeutic effect.

Gene therapy products work by introducing Genetic materialrefers to materials that play a fundamental role in determining the structure and nature of a cell; these include the nucleus, mitochondria, and cytoplasm

See glossary for more terms > into the nucleus of the cell. In order to introduce the genetic material, scientists need a delivery system that can transport the Geneinstructions made of DNA used to create the proteins the body needs to function

See glossary for more terms >, Nucleasean enzyme that divides nucleic acid into nucleotides and other products

See glossary for more terms >, or Short hairpin RNA (shRNA)an artificial RNA molecule that enables gene supression

See glossary for more terms > to the nucleus of a human cell. The vehicle that carries this genetic material is known as a Vectora delivery system used to introduce genetic material into the nucleus

See glossary for more terms >. Like a microscopic delivery truck, the vector can transport and deliver genetic material to target cells or locations in the person’s genome.^{1, 2}

There are two types of vectors, Viral vectora way to deliver genetic material to a cell using the blueprint of a virus as a guide; it may be used to carry genes and change mutated cells to healthy ones

See glossary for more terms > and Non-viral vectora way to deliver genetic material to a cell that is not based on a virus

See glossary for more terms >. Viral vectors are currently a delivery vehicle used in FDA-approved gene therapies. Non-viral techniques are currently being studied as a safe and effective way to deliver genetic material to cells for therapeutic effect.^{1, 3, 4}

Viral and non-viral vectors

Viral vector gene delivery has been utilized in a number of gene therapies due to the virus’ natural ability to access the cells of the body.^1,5

Non-viral vectors are currently being evaluated for long-term expression of the therapeutic genetic material. The most actively researched non-viral vectors include Chemical disruption vectora type of vector that is typically designed to target specific cells and increase the delivery of genetic material to cytosol or nucleus

See glossary for more terms >, Electroporationthe use of an electric field to make a cell more permeable which allows the delivery of genetic material

See glossary for more terms >, and Polymer-based vectorpolymers are one of the substances used to create chemical vectors. These complexes protect DNA and facilitate cell uptake and intracellular delivery

See glossary for more terms >.⁴

Why are viruses used to deliver gene therapy?

Viruses are used as models or blueprints to create viral vectors. This is because viruses are good at entering the nucleus and delivering instructions to a host cell—much like a delivery truck delivers packages to people. Scientists have actually mapped the complete genetic material (genome) of many viruses. They are able to isolate the helpful elements of a virus’ genome—the delivery truck components—and create them on their own as the starting point for a viral vector. To create a viral vector, only a few parts of the virus are used. These parts alone are not adequate to cause viral infection.

Viruses are used as models or blueprints to create viral vectors. This is because viruses are good at entering the nucleus and delivering instructions to a host cell—much like a delivery truck delivers packages to people. Scientists have actually mapped the complete genetic material (Genomethe entire set of genetic instructions found in a cell nucleus

See glossary for more terms >) of many viruses. They are able to isolate the helpful elements of a virus’ genome—the delivery truck components—and create them on their own as the starting point for a viral vector. To create a viral vector, only a few parts of the virus are used. These parts alone are not adequate to cause viral infection.¹

While a number of viral blueprints exist, the choice is based on characteristics such as duration of Gene expressionwhen the information encoded in a gene is used and expressed as an effect or trait

See glossary for more terms >, packaging capacity, target cells, and Immunogenicitythe degree to which a substance triggers an immune response

See glossary for more terms >.¹ Before they can be used, Viral vectora way to deliver genetic material to a cell using the blueprint of a virus as a guide; it may be used to carry genes and change mutated cells to healthy ones

See glossary for more terms > are reviewed by the FDA.^{1, 6}

The final viral vector is like a delivery truck that has updated its contents. The delivery truck just needs the correct address to deliver the new load. Once the genetic material carried by the vector has been delivered to the host cell/tissue, it can help deliver the transgene (or gene of interest) to the nucleus. Once in the nucleus, the transgene can provide instructions to produce the essential functional protein that is needed. After delivery, they are naturally degraded by the host cell/tissue.¹

Gene-ius Questions

Can viral vectors used in gene therapy cause viral infection?

In order for a virus to cause disease, its genome must be nearly complete. When using any virus (adenovirus, adeno-associated virus, or lentivirus) as the basis of a vector for gene therapy, scientists use only certain parts or components of the virus. Before a virus can be used as the basis of a vector, scientists must make a blueprint of the viral genome. They use that blueprint to build the pieces of the virus that are needed to deliver therapeutic genetic material to cells (wild type virus is not used to make a vector). The parts of the viral genome that are not needed for delivery of genetic material are never used. This is so that the vector does not produce the infection that would be caused by the complete virus. Here's an example of how a vector is created from a virus: Let's use a lentiviral vector as our example. This vector is built using a lentivirus (e.g. HIV; most well characterized lentivirus) as the blueprint. The HIV genome is made up of 9 genes and every single gene is required to cause disease. To make the vector, scientists select 3 or 4 different genes from the blueprint of the viral genome. Because only 3 of the 9 genes from the viral genome are created to make the vector, the genome of the virus is not complete and hence, cannot produce the infection caused by the complete virus.

In order for a virus to cause disease, its genome must be nearly complete. When using any virus (adenovirus, adeno-associated virus, or lentivirus) as the basis of a vector for gene therapy, scientists use only certain parts or components of the virus.⁷

Before a virus can be used as the basis of a vector, scientists must make a blueprint of the viral genome. They use that blueprint to build the pieces of the virus that are needed to deliver therapeutic genetic material to cells (Wild Type Virusthe naturally occurring, non-mutated strain of a virus

See glossary for more terms > is not used to make a vector). The parts of the viral genome that are not needed for delivery of genetic material are never used. This is so that the vector does not produce the infection that would be caused by the complete virus.⁷

Here's an example of how a vector is created from a virus:

Let's use a lentiviral vector as our example. This vector is built using a lentivirus (e.g. HIV; most well characterized lentivirus) as the blueprint. The HIV genome is made up of 9 genes and every single gene is required to cause disease.⁷

To make the vector, scientists select 3 or 4 different genes from the blueprint of the viral genome. Because only 3 of the 9 genes from the viral genome are created to make the vector, the genome of the virus is not complete and hence, cannot produce the infection caused by the complete virus.⁷

Viral vectors in gene therapy

Next Level Knowledge

Adenoviral vectors

The first viral vector used in gene therapy was based on adenovirus, which is a virus that causes the common cold. However, some adenoviral vectors (AdV) were found to trigger strong, potentially dangerous, immune reactions in patients. Further research in the use of AdV is being explored.^{1, 8}

Adeno-associated viral vectors

Preparations and use of the adenovirus led to the discovery of adeno-associated viruses (AAV) in the 1960s. AAV consists of a small, single-stranded DNA genome that can be produced in many different variations to focus on certain cell types. Research using AAV-based viral vectors did not gain traction until about 2012.¹

Vectors based on the blueprint of AAV deliver genetic material that nests in the cell nucleus and does not permanently integrate into the cell DNA. This means that the vector replicates outside the host genome and cannot pass down changes to daughter cells during cell division.^{1, 9-11}

AAVs have a smaller genetic packaging capability compared to other viral vectors, which means that they work for some, but not all, targets for gene therapy. As a point of reference, the average packaging capacity for AAV vectors is ~4.5 kb, whereas lentivirus and herpes are 8 kb and ≥30 kb, respectively.¹²

Lentiviral vectors

Lentiviral vectors (LVV) were first explored in gene therapy in the 1980s and became a viable gene therapy technology that evolved quickly in the mid-1990s. Lentiviruses are a species of retrovirusa virus that uses RNA as its genetic material; when a retrovirus infects a host cell, the RNA converts into DNA, which then incorporates into the genome of the host cell

See glossary for more terms >, and the most characterized lentivirus is the human immunodeficiency virus (HIV). Lentiviruses insert their genetic material into dividing and non-dividing cells, integrating the genetic material into the host genome, allowing for continued expression.^1,13

Scientists create lentiviral vectors using the blueprint of a lentivirus (such as HIV-1) because of how effective viruses are at accessing host cells. Lentiviral vectors are mainly used outside the body in ex vivo applications, such as hematopoietic stem cell transplants or engineered T cell therapy. The resulting modified cells are then infused into the patients.¹

Vectors used in gene therapy clinical trials

Chart courtesy of John Wiley and Sons, Journal of Gene Medicine; 2018.¹⁴

Nonviral vectors in gene therapy

A nonviral vector is a delivery mechanism that doesn't use a virus as a blueprint or plan. There are 2 main ways nonviral vectors and their ability to deliver genetic material to the cell are being studied: physically and chemically. Physical methods allow researchers to target cells and deliver genetic material to wherever they want it (think of it like using an injection needle). Chemical methods allow researchers to create cell-specific functionality that affects specific cells through the design of chemical vectors.^{4, 15}

Below, we share 1 example of each type of nonviral delivery method—1 physical (electroporation) and 1 chemical (polymer-based).^{4, 15}

Electroporation is a nonviral delivery method being explored for gene therapy. Typically, electroporation starts with injecting DNA into the target tissue and then using 2 electrodes to administer a series of electric pulses. These pulses cause a pore to form in the cell membrane, through which the DNA can enter the target cells.^{4, 15}

Getting access to target tissue with electrodes can be difficult depending on location. Target cells can be taken out of the body and the electroporation process can happen in a laboratory. This process is called in vitro electroporation.¹⁶

In polymer-based vectors, cationic polymers (polymers with positively charged ions) are mixed with DNA to form complexes called polyplexes. These polyplexes protect the DNA and facilitate uptake and delivery of the DNA into the cell. This chemical nonviral delivery method is being studied in vivo.^{4, 15}

Nonviral vectors may facilitate long-term expression of therapeutic genetic material, although scientists have yet to prove that they can provide long-term treatment effects. Nonviral vectors have become part of clinical trials and continue to be evaluated for use in gene therapy.^{4, 15}

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References

1. STAT Reports. The STAT guide to viral vectors, the linchpin of gene therapy. STAT News; 2019. 2. Zhou H, Zeng X. Energy profile and secondary structure impact shRNA efficacy. BMC Genomics. 2009;10(Suppl 1):S9. 3. Kim TK, Eberwine JH. Mammalian cell transfection: the present and the future. Anal Bioanal Chem. 2010;397(8):3173-3178. 4. Al-Dosari MS, Goa X. Nonviral gene delivery: principle, limitations, and recent progress. AAPS J. 2009;11(4):671-681. 5. Collins M, Thrasher A. Gene therapy: progress and predictions. Proc Biol Sci. 2015;282(1821):20143003. 6. Food and Drug Administration. Testing of retroviral vector-based human gene therapy products for replication competent retrovirus during product manufacture and patient follow-up. Guidance for industry. Accessed March 1, 2020. https://www.fda.gov/vaccines-blood-biologics/guidance-compliance-regulatory-information-biologics/biologics-guidances 7. AIDSinfo. HIV/AIDS Glossary. Accessed June 9, 2020. https://aidsinfo.nih.gov/understanding-hiv-aids/glossary/753/wild-type-virus 8. Wold WS, Toth K. Adenovirus vectors for gene therapy, vaccination and cancer gene therapy. Curr Gene Ther. 2013;13(6):421-433. 9. Colella P, Ronzitti G, Mingozzi F. Emerging issues in AAV-mediated in vivo gene therapy. Mol Ther Methods Clin Dev. 2017;8:87-104. 10. Henckaerts E, Linden RM. Adeno-associated virus: a key to the human genome? Future Virol. 2010;5(5):555-574. 11. Thomas CE, Ehrhardt A, Kay MA. Progress and problems with the use of viral vectors for gene therapy. Nat Rev Genet. 2003;4(5):346-358. 12. Sheridan C. Gene therapy finds its niche. Nat Biotechnol. 2011;29(2):121-128. 13. Durand S, Cimarelli A. The inside out of lentiviral vectors. Viruses. 2011;3(2):132-159. 14. Gene therapy clinical trials worldwide. Journal of Gene Medicine. John Wiley and Sons, Ltd. 2019. http://www.abedia.com/wiley/vectors.php. Accessed March 24, 2020. 15. Ramamoorth M, Narvekar A. Non viral vectors in gene therapy – an overview. J Clin Diagn Res. 2015;9(1):GE01-6. 16. Morgan RA, Gray D, Lomova A, et al. Hematopoietic stem cell gene therapy—progress and lessons learned. Cell Stem Cell. 2017;21(5):574-590.