One offshoot of studies with transgenic animals has been the advancement of medical treatments for human diseases that involve manipulating genetics, rather than administering drugs. The ap plications that show the most promise are gene therapy, which aims to replace a nonfunctional gene with its normal counterpart, and regulatory RNAs, which manage DNA expression of abnormal genes.
Gene Therapy
We have known for decades that for certain diseases, the disease phenotype is due to a missing or nonfunctioning protein. Often referred to as “loss of function” diseases, examples include cystic fibrosis, hemophilia, sickle-cell anemia, some forms of dwarfism, and muscular dystrophy. A few of these diseases can be treated by administering the correct protein—recall the use of recombinant growth hormone to treat pituitary dwarfism. But in the majority of cases, a replacement protein is not yet available. About 30 years ago, medical geneticists began to explore the possibility of restoring function by means of gene therapy, by inserting the normal functioning gene into the cells of afflicted patients.
The goal of gene therapy would be to permanently cure the physiological dysfunction by repairing the genetic defect. There are two strategies for this therapy. In ex vivo therapy, the normal gene is cloned in vectors such as adenoviruses or retroviruses that have been modified to carry a corrective gene into cells of the patient. Tissues removed from the patient are incubated with these genetically modified viruses to transfect them with the normal gene. The transfected cells are then reintroduced into the patient’s body by transfusion (process figure 1). In contrast, the in vivo type of therapy skips the intermediate step of incubating excised patient tissue. Instead, the naked DNA or a virus vector is directly introduced into the patient’s tissues.
Process Figure 1 Ex vivo gene therapy. (1) Stem cells are removed from the bone marrow of a patient with a genetic defect. (2) A normal version of the gene is isolated from a healthy subject, cloned, and inserted into a viral vector. (3) The virus is used to infect the patient’s cells in the laboratory. The viral vector inserts the healthy version of the gene into the genome of the patient. (4) The transfected cells are infused into the patient, where they move to the bone marrow. The transfected cells will continue to multiply in the bone marrow, producing protein from the corrected gene.
Clinical testing using various types of gene therapy is per formed on human volunteers with a diagnosed genetic disorder. Over 2,000 of these trials have been or are being implemented in the United States and other countries. Most trials target cancer, single-gene defects, and infections. The most dramatic example of this type of treatment was seen in the very first gene therapy trial conducted in 1990. In this case, two girls suffering from adenosine deaminase (ADA) deficiency, a fatal immunologic disorder caused by a missing enzyme, received infusions of their own hematopoietic stem cells that had been transformed with a healthy copy of the adenosine deaminase gene.
As with most medical experimentation, some of the earlier gene therapy trials experienced setbacks. A serious problem arose with the use of the viral vectors. Even though they were supposed to be “harmless,” five children infused with a recombinant mouse retrovirus developed leukemia when the vector DNA integrated at a cancer-inducing site on a chromosome. After this event, researchers worked to develop modified adeno-associated viral vectors to greatly reduce the chances of this happening.
Over the past several years, medical geneticists reported major successes in curing 20 children born with the devastating genetic disease termed X-1-linked SCID (severe combined deficiency disease). These children lacked a functioning immune system from birth, which researchers restored by inserting copies of the functioning gene into their stem cells using a safer recombinant virus. Two giant strides in gene therapy have been made in the last few years. In 2018, the FDA approved Luxturna, a gene therapy for a progressive form of blindness called Leber congenital amaurosis. The treatment uses a genetically engineered virus to deliver a healthy gene, designated RPE65, directly to the retina after doctors inject the virus into the eyeball itself. The protein product of the gene is an enzyme that nourishes the cells of the retina; many of the children in the clinical trial reported immediate and dramatic improvement in their vision. In 2019, Zolgensma was approved to cure spinal muscular atrophy (SMA), a group of debilitating neuromuscular disorders that is a leading genetic cause of death in infants. The disorder is caused by lack of a working SMN1 gene, and the treatment uses an adenovirus capsid to deliver a functional SMN1 gene to the nucleus of motor neurons. Zolgensma has the dubious distinction of being the most expensive medication on earth, as a one-time dose costs $2.125 million.
Therapy with Regulatory RNAs
Tens of thousands of genetic disorders have been described. Most are due to nonfunctional or missing genetic material, but a few of them result from the expression of abnormal, mutated genes. These conditions, termed “gain of function” diseases, include a number of cancers, some forms of Alzheimer’s disease, Huntington’s dis ease, and fragile X syndrome. Since every cell in the body carries these faulty but active genes, merely replacing them is not yet an option.
For these types of diseases, genetic researchers are taking a lesson from the natural strategies used by bacteria and eukaryotic organisms for regulating gene expression. Recall that these organisms use regulatory RNA or RNAi, including small interfering RNAs and microRNAs, to silence gene expression. This knowledge has driven the development of genetic-based drugs that interfere with mRNA processing and protein synthesis. Some of the most promising therapies involve the targeting of RNA virus genomes by small RNAs. They have been used to degrade HIV and respiratory syncytial viruses. They have also been tested as a treatment for macular degeneration and as a therapy to silence cancer genes.
In late 2018, the FDA approved the first RNAi therapy, Onpattro, to treat the deadly genetic disorder hereditary transthyretin mediated amyloidosis. A great deal of research was directed toward the development of lipid nanoparticles used to deliver the interfering RNA to the proper tissue in the body. This knowledge proved invaluable just two years later when similar nanoparticles were used to deliver the mRNA-based SARS-CoV-2 vaccines. Because the RNAi agent can be targeted to a specific sequence of DNA, side effects are rare. The biggest obstacles involve how to deliver the RNAi drugs into deep tissues where their targets lie and the fact that these drugs need to be taken for life. An annual course of Onpattro is $450,000.
