Gene therapy works by altering the genetic code to recover the functions of critical proteins. Proteins are the workhorses of the cell and the structural basis of the body’s tissues. The instructions for making proteins are carried in a person’s genetic code, and variants [or mutations] in this code can impact the production or function of proteins that may be critical to how the body works. Fixing or compensating for disease-causing genetic changes may recover the role of these important proteins and allow the body to function as expected.
Gene therapy can compensate for genetic alterations in a couple different ways.
- Gene transfer therapy introduces new genetic material into cells. If an altered gene causes a necessary protein to be faulty or missing, gene transfer therapy can introduce a normal copy of the gene to recover the function of the protein. Alternatively, the therapy can introduce a different gene that provides instructions for a protein that helps the cell function normally, despite the genetic alteration.
- Genome editing is a newer technique that may potentially be used for gene therapy. Instead of adding new genetic material, genome editing introduces gene-editing tools that can change the existing DNA in the cell. Genome editing technologies allow genetic material to be added, removed, or altered at precise locations in the genome. CRISPR-Cas9 is a well-known type of genome editing.
Genetic material or gene-editing tools that are inserted directly into a cell usually do not function. Instead, a carrier called a vector is genetically engineered to carry and deliver the material. Certain viruses are used as vectors because they can deliver the material by infecting the cell. The viruses are modified so they can't cause disease when used in people. Some types of virus, such as retroviruses, integrate their genetic material [including the new gene] into a chromosome in the human cell. Other viruses, such as adenoviruses, introduce their DNA into the nucleus of the cell, but the DNA is not integrated into a chromosome. Viruses can also deliver the gene-editing tools to the nucleus of the cell.
The vector can be injected or given intravenously [by IV] directly into a specific tissue in the body, where it is taken up by individual cells. Alternately, a sample of the patient's cells can be removed and exposed to the vector in a laboratory setting. The cells containing the vector are then returned to the patient. If the treatment is successful, the new gene delivered by the vector will make a functioning protein or the editing molecules will correct a DNA error and restore protein function.
Gene therapy with viral vectors has been successful, but it does carry some risk. Sometimes the virus triggers a dangerous immune response. In addition, vectors that integrate the genetic material into a chromosome can cause errors that lead to cancer. Researchers are developing newer technologies that can deliver genetic material or gene-editing tools without using viruses. One such technique uses special structures called nanoparticles as vectors to deliver the genetic material or gene-editing components into cells. Nanoparticles are incredibly small structures that have been developed for many uses. For gene therapy, these tiny particles are designed with specific characteristics to target them to particular cell types. Nanoparticles are less likely to cause immune reactions than viral vectors, and they are easier to design and modify for specific purposes.
Researchers continue to work to overcome the many technical challenges of gene therapy. For example, scientists are finding better ways to deliver genes or gene-editing tools and target them to particular cells. They are also working to more precisely control when the treatment is functional in the body.
Scientific journal articles for further reading
Bulcha JT, Wang Y, Ma H, Tai PWL, Gao G. Viral vector platforms within the gene therapy landscape. Signal Transduct Target Ther. 2021 Feb 8;6[1]:53. doi: 10.1038/s41392-021-00487-6. PMID: 33558455. Free full-text article from PubMed Central: PMC7868676.
Duan L, Ouyang K, Xu X, Xu L, Wen C, Zhou X, Qin Z, Xu Z, Sun W, Liang Y. Nanoparticle Delivery of CRISPR/Cas9 for Genome Editing. Front Genet. 2021 May 12;12:673286. doi: 10.3389/fgene.2021.673286. PubMed: 34054927. Free full-text article from PubMed Central: PMC8149999.
Only a handful of the hundreds of known vertebrate retroviruses have been deliberately subverted for use as carriers of recombinant genetic material. Retroviruses receive their name from the fact that their genome undergoes conversion from RNA to DNA following infection of a host cell. Also characteristic of retroviruses and uncommon for most other types of viruses is that the genome of the retrovirus integrates itself permanently into the DNA of the host cell. Once integrated into the host genome, the inserted provirus acts as a factory for producing more retroviral RNA genomes and expressing retroviral packaging proteins. Both components combine to form viral particles that bud from the surface of the infected cells.
For gene transfer to mammalian cells, most recombinant retroviral vectors are derived from the Moloney murine leukemia virus [MLV], a mammalian type C retrovirus.1Recently, recombinant human immunodeficiency virus [HIV] has also been used to transfer recombinant genetic material to mammalian cells and tissues. Both viruses have their individual strengths and weaknesses as gene transfer vectors. Moloney murine leukemia virus genome that encodes the canonical group-specific core antigen [Gag], polymerase [Pol], and envelope [Env] gene products is relatively well understood and has been genetically engineered to produce both packaging cell lines and plasmids for the production of recombinant virus. The MLV is also attractive for reasons of safety, and while there are exceptions, neither the production of recombinant MLV-based retroviruses nor cells infected with these viruses are generally considered high-risk biohazards.
Human immunodeficiency virus–based vectors are capable of infecting nondividing, postmitotic cell types that include neurons, whereas MLV-based vectors are only able to efficiently infect actively dividing cells. This characteristic, and extended high-level gene expression, makes them ideal therapeutic agents for the transfer of novel genetic material to the adult central nervous system [CNS]. The weakness of HIV-based vectors, however, is that they currently require stringent biosafety procedures to produce safe recombinant virus. Furthermore, the presence of an infectious lentivirus [HIV] in the human population warns that measures need to be taken to ensure that the recombinant virus is not rescued by wild-type infection of human subjects. Research is currently directed at the production of novel recombinant retroviruses that will combine the efficiency and flexibility of HIV with the safety and simplicity of MLV-like retroviral vectors.
The production of recombinant retroviruses
All viral methods of gene transmission rely on separation of the means of forming an infectious particle and the infectious particle itself. A virion may be separated into 2 components: the nucleic acid genome that encodes the proteins necessary for perpetuation of the viral life cycle, and the protein package that houses and conveys the genome. Development of recombinant retroviral vectors began with plasmids encoding contiguous retroviral genomes with both noncoding and coding regions [Figure 1, A]. The noncoding regions include the retroviral long terminal repeats that function as promoters for the expression of viral proteins and other packaging signals [ψ] required for encapsidation, reverse transcription, and insertion into the host genome. The coding regions encode Gag, Pol, and Env polyproteins required for production of infectious virions. The regions of DNA encoding the Gag, Pol, and Env proteins were excised from the intact retroviral genome, leaving noncoding, but essential retroviral sequences [Figure 1, A]. The excised gag, pol,andenvgenes were then introduced into cultured cells to create packaging cell lines. In 1983, Mann et al2demonstrated that stable transduction of plasmids carrying the noncoding vector components [long terminal repeats, etc] into packaging cells results in the production of viral particles that are able to infect target cells but are themselves unable to replicate or repackage into new infectious viral particles.
Foreign genes are introduced into retroviral vectors in the vacancy left by gag, pol,and envgene deletion from the complete genome [Figure 1, A]. The insertion of genes encoding selectable markers, internal promoters, and multiple cloning sites has converted currently available retroviral constructs into complex, multifunctional vectors for gene transfer. The stable introduction of a recombinant retroviral vector into a packaging cell line results in what is commonly referred to as a "producer cell line." Producer cell lines often produce replication-defective retroviruses at titers greater than 1×105infectious particles per milliliter.
Recently, an alternative to the production of recombinant retroviruses using packaging cells and stable producer cell lines is to transiently package virus using cotransfection of plasmids encoding the packaging proteins and the retroviral vector simultaneously into easily transduced cell types [Figure 1, B]. Although the transfected cells produce virus for only a few days, within this time they can produce titers exceeding the capacity of traditional producer cell lines. The transiently higher titer stems from both the very high plasmid copy number [>10-50 copies] in transfected cells relative to the lower copy number characteristic of stable producer cells [