Rewriting Fate

PN dna landscapes

Gene Editing as Tool & Treatment

by Sver Aune

For some patients, genetic disorders can seem written into DNA as permanently as eye color. Yet defective genes and their attendant symptoms could be quelled with gene therapy.

Laboratory techniques to write over faulty genes typically use pieces of viruses called vectors. Viral vectors infect diseased cells with the functional human gene, which is then replicated alongside the defective one. In order to avoid immune reactions, the ability of the virus to replicate itself is first neutralized. But past success in experiments did not guarantee success in clinical trials. Gene therapy research was shaken in 1999 when an 18-year-old man in a clinical trial died from an overreaction to the virus carrying the therapeutic gene for his condition.1

That experience transformed the field. Since 1999, gene therapies have improved greatly due to intensive preclinical research. Viral vectors have been modified to more accurately target only diseased cells. Novel methods such as CRISPR enable researchers to remove a defective gene entirely through genome editing. At MUSC, researchers are conducting new clinical trials and original research that give hope to patients with genetic disorders.

Our cells, ourselves

A genetic mutation affects the cells or organs that need the protein encoded by that gene. A pair of high-profile clinical trials now underway at MUSC are testing therapies for mutations in genes that encode blood proteins. The goals are to determine how to guide the functional gene to the cells that need it and how much encoded protein is needed to offset the mutation.


MUSC is a site for a new phase 1/2 clinical trial of safety and efficacy for a first-in-man gene therapy to treat the lung and liver disease alpha-1 antitrypsin deficiency (Alpha-1). Charlie B. Strange, M.D., professor of medicine in the Division of Pulmonary, Critical Care and Sleep Medicine, is principal investigator on the trial and director of the national Alpha-1 Foundation Research Registry.

The new therapy is administered to combat the development of emphysema in patients as young as thirty by more efficiently boosting alpha-1 antitrypsin protein (A1AT) levels in the lungs. A1AT is the second-most abundant protein in the blood and protects lung epithelial cells from environmental toxins such as cigarette smoke, according to Strange. Alpha-1 patients have about 15 percent of the normal level of A1AT due to a rare genetic mutation that prevents A1AT from folding correctly in the liver. To prevent early-onset lung disease, patients require weekly intravenous infusions of A1AT for life.2

In the trial, an adeno-associated viral vector (AAV) carrying the normal gene for A1AT is infused into the pleural space that wraps around the lungs. If enough A1AT protein persists in the pleural space, it could halt emphysema progression and improve symptoms.

AAV is not known to cause disease in humans and can sustain replication of the functional DNA it carries into human cells, thereby allaying the symptoms caused by the mutated gene. However, AAV works in only a minority of the human cells it contacts.For example, in the first clinical studies using AAV to deliver the gene for A1AT to the leg muscles of patients, only a fraction of muscle cells produced A1AT protein. As a result, protein levels in the bloodstream rose, but not nearly enough to change the course of the disease.2

The trial is testing pleural infusion versus intravenous infusion of a viral vector (Adverum Biotechnologies). In preclinical studies, more A1AT was made in the lungs when the vector was infused into the pleural space than when it was injected through the bloodstream. The trial is being conducted at MUSC and Temple University.

The ultimate goal is to achieve complete remission of symptoms while freeing Alpha-1 patients from weekly infusions.

“We want to get gene therapy that will be given once and cure your symptoms for the rest of your life,” says Strange.

Pushing the envelope to treat sickle cell disease

Julie Kanter, M.D., a hematologist and director of sickle cell research, is an investigator on an international phase 1/2 clinical safety trial — the HGB206 trial — for a new gene therapy for sickle cell disease (SCD). As with Alpha-1 gene therapy, there is hope that the therapy will repair enough red blood cells to compensate for those with the sickle defect. In fact, a 13-year-old patient in a clinical trial in France has experienced complete resolution of symptoms with the therapy.4

Patients with SCD have inherited from both parents a mutation in the gene for hemoglobin that gives red blood cells a sickle-like shape and reduces oxygen delivery throughout the body. SCD can cause episodes of severe pain and complications including stroke. Currently, a stem cell transplant is the only cure.4

The gene therapy in the trial, sponsored by Bluebird Bio, uses a lentiviral vector designed to carry a globin gene that is very similar to normal hemoglobin A with an alteration that makes it even more anti-sickling like hemoglobin F.

“Lentiglobin is the lentiviral vector, which I always think of as the envelope,” says Kanter. “They put a new globin gene in as the letter, and that’s called T87Q.”

Lentiviral vectors are more efficient than AAV at incorporating the DNA they carry into host cells. However, lentiviruses can cause severe disease in humans, limiting their potential use in gene therapy.5

The use of stem cells may solve this problem. Stem cells are removed from a patient’s bone marrow and infected with lentiglobin. The virus is then removed to mitigate an immune response, and the stem cells containing T87Q DNA are transplanted back into the patient’s bone marrow. Those stem cells can then develop into normal red blood cells that make T87Q that counteracts the SCD defect.

There is considerable excitement building around gene therapy for SCD. Yet while 50 percent of the French teen’s red blood cells are making T87Q, the rest are sickled cells.

“While we’re excited about some of the early successes, we have a long way to go,” says Kanter.

Brainstorming new gene therapies

These are just two examples of gene therapies that are moving from time-tested research tool to treatment at MUSC Health. Preclinical work on new gene-editing tools can give us a glimpse at the future of gene therapy in common brain disorders and beyond.

Mapping addiction

At the MUSC Charleston Alcohol Research Center (ARC), scientists are charting addiction in the brain with a unique tool: designer receptors exclusively activated by designer drugs (DREADDs).6 In preclinical models, a given DREADD delivered to specific brain regions can render neurons active or inactive in response to systemic administration of a specially designed drug that is otherwise innocuous. The genes that encode these receptors are delivered to neurons by AAV. Several different types of DREADDs can be used at once, providing a sophisticated level of control called multiplexing. In this way, researchers in the ARC can determine which circuits in the brain are involved in addiction, relapse, and automated behavior.

In the future, patients might pop a pill to calm the addiction circuit of their brain each time a craving arises. Yet there are puzzles to solve before that can happen, according to Howard C. Becker, Ph.D., director of the ARC. For example, it may be difficult to target the many areas of the brain and many genes affected by addiction. Also, the viruses must be injected directly into the brain during neurosurgery.

“Because we don’t have easy access to the brain, that’s a barrier,” says Becker. “But in time and with more research, eventually this could very well be the wave of the future, especially for neuropsychiatric-related illnesses.”

Shining a light on narcolepsy

Another brain research group is modifying the gene suspected in narcolepsy in preclinical tests. Meng Liu, M.D., Ph.D., of the Department of Psychiatry and Behavioral Sciences received R01 funding in 2016 to develop a gene therapy for narcoleptic cataplexy, a condition affecting two-thirds of narcolepsy patients, in which irresistible sleep is triggered by strong emotion. Although the exact cause is unknown, many patients have very low levels of the neuropeptide orexin, which regulates sleep.7 Liu’s research team is following this promising clue in preclinical studies. The group is testing an AAV carrying the gene for orexin. They are combining delivery of the orexin gene and optogenetics, a technique that renders neurons sensitive to tiny lights inserted in the brain. The plan is to map the exact neural pathways that control narcolepsy and, eventually, develop therapies such as orexin gene delivery to treat the disorder.

Latest gene-editing tools

Many researchers at MUSC are embracing innovations in gene-editing tools. One such tool — CRISPR — is considered the future of gene therapy for patients with genetic conditions ranging from Down Syndrome to Alzheimer’s disease to cancer.8 This enthusiasm stems from CRISPR’s power to target any DNA sequence with unparalleled precision. In contrast to viral vectors that compensate for a mutation, the CRISPR system retools an antiviral defense mechanism found in bacteria to remove a faulty gene completely and permanently replace it with the correct one.

CRISPR has become the method of choice for rewriting genes in preclinical models, according to Alexander Awgulewitsch, Ph.D., scientific director of the new transgenic and genome editing core at MUSC. The core opened in May 2017 to meet the growing demand for gene editing among preclinical researchers across the state of South Carolina. The new core uses CRISPR and other tools to help researchers add, subtract or silence genes to better understand disease and look for cures.

Gene therapies are maturing, driven by new research and enthusiasm for their potential to help patients. With new clinical trials and gene-editing tools in the pipeline, there is hope in the medical community that gene therapy will help patients with a genetic disorder rewrite their fate.


1 The Editors.Nature 2016;534:590.

2 Flotte TR, Mueller C. Hum Mol Genet. 2011;20(R1):R87-R92.

3 Nonnenmacher M, Weber T. Gene Ther. 2012;19:649-658.

4 Ribeil J-A, et al. N Engl J Med. 2017;376:848-855.

5 Kajaste-Rudnitski A, Luigi N. Hum Gene Ther. 2015;26(4):201-209.

6 Urban DJ, Roth BL. Annu Rev Pharmacol Toxicol. 2015;55:399-417.

7 Liu M, et al. Eur J Neurosci. 2016;43(5):681-688.

8 Ma Y, et al.FEBS J. 2014;281(23):5186-5193.