Investigators at The Children’s Hospital of Philadelphia are exploring a new gene therapy approach that aims to reactivate the production of fetal hemoglobin as a potential intervention for patients with sickle cell disease. In the U.S., it is estimated that sickle cell disease affects 100,000 newborns, especially Hispanic-Americans and African-Americans.
The inherited disease distorts the red blood cells into a sickle shape, like the letter “C,” that blocks blood flow and damages blood vessels and many organs. A sickle cell gene mutation tells the body to make a defective type of hemoglobin, which is the oxygen transport protein in red blood cells.
Pre-empting the effects of this sickle cell gene mutation has been a focus of CHOP hematology researcher Gerd A. Blobel, MD, PhD, and the August edition of Cell reported his novel findings. Dr. Blobel and his co-authors described how they altered the genetic architecture behind a developmentally controlled process called hemoglobin switching.
A form of hemoglobin that has anti-sickling properties is made only during the fetal period when red blood cells are produced by the liver. Shortly after birth, a transition occurs that silences the fetal globin genes as the bone marrow gradually takes over blood formation. Gene expression shifts to mostly create the adult form of hemoglobin, which in sickle cell disease will be the variant type.
Adult hemoglobin (HbA) almost completely replaces fetal hemoglobin (HbF) within six months after birth, which helps to explain why patients with sickle cell disease do not experience symptoms as newborns. It has been known for a long time that patients with sickle cell disease who have higher ratios of HbF tend to experience a milder course of the disease.
“A major driver in the field for many years has been to understand the molecular basis and the machinery that controls that switch,” Dr. Blobel said. “The goal is ultimately to overcome the silencing of the fetal globin genes and turn them back on.”
His research team’s particular strategy to manipulate gene expression not only elevates the amount of HbF but also downregulates the amount of faulty HbA, thereby reducing the sickle cell inducing properties of the mutated form of HbA.
The current study employs artificial zinc finger protein technology that Dr. Blobel and colleagues adapted for use in hemoglobin regulation and described in Cell two years ago. At the time, the zinc finger proteins were engineered in a way that they locate to specific sites on the chromosomes and foster contacts between chromosomal regulatory elements called promoters and enhancers that reside very far apart on the chromosome. This juxtaposition led to the formation of a chromosomal loop.
“Our 2012 study was the first in which such looped gene contacts were produced at a normal gene in its native location,” Dr. Blobel said. “We thought, now that we have this useful system, let’s explore whether it can be put to therapeutic use.”
In the present study, the researchers showed that they can use forced gene looping to override the stringent developmental gene expression of the globin gene cluster, which is composed of embryonic, fetal, and adult type genes responsible for the creation of hemoglobin. During development, a powerful enhancer for the expression of all these genes, called the locus control region (LCR), physically contacts the embryonic, then fetal, and later the adult genes via chromatin looping.
Dr. Blobel’s team designed zinc fingers in a way that they would promote looped contacts between the LCR and the fetal genes in adult red blood cells. This approach worked well and indeed enhanced the expression of fetal genes and reduced the level of the adult type genes. In the context of sickle cell disease, this would be a double benefit since both high fetal gene expression and low levels of the mutated toxic form of adult hemoglobin would ameliorate the disease.
“This is a novel way to manipulate gene expression via altering chromatin architecture,” Dr. Blobel said.
The researchers carried out the experiments in cultured blood cells from mice and humans. The next step is for the team to use a pre-clinical model to further test their strategy. This involves the use of genetically engineered mice bearing the human globin genes (including the sickle cell anemia gene mutation) in place of the mouse genes. These animals have manifestations of sickle cell disease similar to human patients. Blood stem cells from these mice will be modified with a viral gene transfer vector that expresses the zinc-fingered looping protein, and then used in bone marrow transplants to see if it can correct the disease in the animal model. If this is successful, the long-term goal is to start a clinical trial in humans.
The Sickle Cell Center at CHOP has a longstanding commitment to sickle cell disease research and is one of the largest programs of its kind in the U.S., providing comprehensive care to almost 1,000 patients. Dr. Blobel’s work is supported by funding from the National Institute of Diabetes Digestive and Kidney Diseases and the National Heart, Lung, and Blood Institute.