The concept of putting genes into the human body to correct missing or dysfunctional areas of the genome first emerged in the 1960s. Since then, the field of gene therapy has experienced groundbreaking research discoveries, tragic pitfalls, and finally, a resurgence in interest and a rise in breakthroughs.
Explore this interactive Science Milestone from Drug Discovery News to learn about the complicated past of early gene therapy discoveries and the technologies that eventually led to gene therapy success.
Milestone
Putting Genes into the Human Body
By Maggie Chen
The concept that specific changes in the genome caused diseases was an intriguing idea to many scientists in the 1960s and 70s. Because some diseases traced to a missing or malfunctioning gene, scientists designed therapies to target defective regions of the genome. These therapies, categorized as gene therapies, have emerged over the past few decades as potential strategies for treating underlying disease mechanisms rather than alleviating disease symptoms.
1960s-1980s Putting genes into cells
In the early 1960s, several research groups demonstrated that injecting cells with DNA could yield gene expression changes. The idea of injecting wild type genes into cells to replace abnormal disease-associated genes was tantalizing. However, scientists needed to find a way to stably introduce new gene copies into the body. In the 1970s, scientists leveraged the natural cell-penetrating abilities of retroviruses or the passive cytoplasmic transfer of plasmids to deliver genetic sequences into mammalian cells.
In 1979, Martin Cline, a scientist at the University of California, Los Angeles (UCLA), introduced the human hemoglobin gene into mouse bone marrow cells using bacterial plasmids. The therapy partially reconstituted the bone marrow in mice that had been treated with irradiation (1). The next year, Cline tested this therapy in a human trial. He and his team took bone marrow cells from two patients with severe betathalassemia, a disease caused by one mutated copy of the gene that produces hemoglobin, transfected those cells with plasmids containing a wild type copy of the human hemoglobin gene, and then infused those cells back into the patients (1).
Because Cline did not receive permission from the UCLA institutional review board, the trial received widespread criticism. Cline lost his chair position and government funding, catalyzing a needed discussion about the ethics, technicalities, and rules for gene therapy. This led to the creation of several scientific oversight committees (1).
1990s An initial gene therapy trial
While Cline’s gene therapy trial alerted the scientific community to the career penalties and potential dangers to patients of performing unregulated gene therapy research, scientists continued to pursue approved, safer, and more effective ways of delivering genes to patients. In 1990, a group of scientists at the National Institutes of Health began the first FDA-approved clinical trial for gene therapy. In this trial, scientists treated two young patients with severe combined immunodeficiency (SCID), who lacked the adenosine deaminase (ADA) gene. “I got involved to find a therapy to treat kids who were going to die and didn’t have any other therapy that was effective,” said Michael Blaese, a retired immunologist previously at the National Cancer Institute who co-led the trial. Along with several geneticists, cancer surgeons, and biologists, Blaese developed a method for isolating T lymphocytes from patients. He and his team transfected these T lymphocytes with a retroviral vector containing a copy of the ADA gene, expanded the cells into a larger population, and then transfused them back into the patients (2). “The plan was to give infusions to the kids about every two months for the first year and then look in their circulating cells to see if the ADA gene was there,” Blaese explained. During the gene therapy trial, the two patients received a noncurative treatment that helped alleviate SCID symptoms but did not solve their immune deficiency. Over the course of four years, immunoglobin production significantly increased in the two patients, indicating that the gene therapy contributed to a more robust immune response (2). Years after the therapy, the edited T lymphocytes from those infusions persist. “[The patients] still have circulating cells that we gave them 30 years ago,” said Blaese.
2000s RNAi enters the field
While the idea of injecting genes into patients to create new functional proteins gained traction, 2001 brought about a new method for regulating protein expression: small interfering RNA (siRNA), which can change different pathways involved in RNA interference (RNAi) (3). RNAi is a natural biological process during which certain RNA molecules bind to specific genomic sequences to suppress gene expression. With RNAi, scientists planned to squash the expression of a faulty gene rather than produce a new protein by introducing a new gene.
“We were interested in trying to develop a treatment for viral hepatitis,” said Mark Kay, a geneticist at Stanford University. With its capability to silence disease-causing genes, RNAi seemed like a good option.
In 2002, Kay’s group demonstrated in mice that siRNA designed to bind to the hepatitis C virus suppressed expression of a gene fused with a portion of the virus (4). Then in 2003, Kay’s group demonstrated that similar RNAi constructs called short hairpin RNA (shRNA), successfully inhibited hepatitis B viral replication in a mouse model (5).
While this research demonstrated the promise of leveraging RNAi to treat disease, Kay soon learned that this technology required careful modulation (6). In 2006, he and his team gave mice a high shRNA dose. “We knocked [the gene] down, but then the animals started going into liver failure,” reflected Kay. This suggested that turning RNAi into a successful therapeutic would require a more tailored delivery approach to optimize the quantity of RNAi constructs and to yield results with minimal side effects.
2010s AAV-based gene therapy
On the quest to find the ideal method to deliver functional genes or modify gene expression, some scientists turned to adeno-associated viruses (AAV). In the 1980s, scientists discovered that AAV could deliver genes into human cells and target specific organs such as the brain or liver. Additionally, the ability of AAV to deliver DNA instead of RNA made them attractive alternatives to retroviral vectors used in the past. But several early AAV-based gene therapies in the 1990s failed due to severe immune reactions or cancer caused by unanticipated AAV integration near oncogenes that resulted in several patient deaths. These early tragedies caused scientists to place greater emphasis on clinical trial monitoring, nonclinical animal studies, and potential side effects of viral-based gene therapies.
In the 2010s, researchers carefully attempted to leverage AAV for gene therapy again. In 2013, Robert Maclaren, an ophthalmologist at the University of Oxford, developed an AAV-based therapy to treat choroideremia, a genetic disease that leads to vision loss. People with choroideremia lack the choroideremia (CHM) gene, so Maclaren and his team designed their AAV vector to deliver a functional copy of the CHM gene to patients.
In 2014, the team reported positive findings from an initial Phase1/2 clinical trial (7), which led to several other publications in 2016 and 2018 where they reported that their AAV-based therapy provided long-term vision improvements in patients with choroideremia (8,9). Despite these positive findings, Maclaren noted that there were still areas for improvement. “Immunogenicity, in my view, is the single biggest challenge we have at the moment with AAV,” said Maclaren. “It’s difficult to predict because it’s different in different people.” Using AAV, Maclaren went on to develop Luxturna, a gene therapy for inherited blindness. In 2017, Luxturna became the first FDA-approved gene therapy in the United States, opening the door for the development and approval of a host of other gene therapies over the next few years. Maclaren is still working to develop an AAV-based gene therapy for choroideremia, and a Phase 3 clinical trial for the therapy is underway.
2012-present CRISPR and the epigenome
In 2018, the first RNAi-based drug was approved by the FDA. As more RNAi and AAV-based gene therapies traveled further into the FDA regulatory approval pipeline, CRISPR-Cas9 methods emerged in 2012. This technology enabled scientists to make specific genome edits to produce effects ranging from modulating gene expression to changing specific bases in DNA or RNA.
In 2020, the first systemic CRISPR-Cas9 therapy targeting transthyretin (TTR) amyloidosis, a disease where misfolded TTR protein accumulates in tissues and causes organ dysfunction, entered clinical trials (10). CRISPR-based clinical trials for sickle cell disease, various cancers, and HIV have also received the green light from the FDA.
For Maclaren, CRISPR represents a powerful new tool in the suite of strategies for gene therapy. “Everyone in my research team is working on CRISPR,” he said. “What we’re doing is using the AAV platform as a delivery system for gene editing technology.”
Combining decades of gene therapy research with emerging gene editing technologies has led to several interesting developments. Kay’s group recently found that AAV capsids or their protein shells play critical roles in mediating the epigenome. “It turns out that the capsid is helping direct the epigenetic state of the [host] genome once it gets into the cell,” he said. This suggests that optimizing AAV design could increase the efficacy of delivered genetically-modified genes for gene therapy.
“I couldn’t be happier to see the technologies coming along that make it possible to treat the diseases I was concerned about for all those years,” Blaese said. “When I first went to the NIH, I went to a lot of funerals. Now, I get to go to weddings.”
REFERENCES
1. Beutler, E. the cline affair. Molecular Therapy 4, 396–397 (2001).
2. Blaese, R. M. et al. T Lymphocyte-Directed Gene Therapy for ADA− SCID: Initial Trial Results After 4 Years. Science 270, 475–480 (1995).
3. Elbashir, S. M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001).
4. McCaffrey, A. P. et al. RNA interference in adult mice. Nature 418, 38–39 (2002).
5. McCaffrey, A. P. et al. Inhibition of hepatitis B virus in mice by RNA interference. Nat Biotechnol 21, 639–644 (2003).
6. Grimm, D. et al. Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature 441, 537–541 (2006).
7. MacLaren, R. E. et al. Retinal gene therapy in patients with choroideremia: initial findings from a phase 1/2 clinical trial. The Lancet 383, 1129–1137 (2014).
8. Edwards, T. L. et al. Visual Acuity after Retinal Gene Therapy for Choroideremia. N Engl J Med 374, 1996–1998 (2016).
9. Xue, K. et al. Beneficial effects on vision in patients undergoing retinal gene therapy for choroideremia. Nat Med 24, 1507–1512 (2018).
10. Gillmore, J. D. et al. CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis. New England Journal of Medicine 385, 493–502 (2021).