Lentiviral cell therapies hold promise for monogenic blood disorders

By Samantha Black, PhD, The Science Advisory Board editor in chief

May 19, 2021 -- Lentiviral vectors show promise for monogenic blood disorders and the future is bright for these genetically modified cell therapies, according to Dr. Michel Sadelain, PhD, who discussed the topic during the George Stamatoyannopoulos Memorial Lecture at the annual meeting of the American Society of Gene & Cell Therapy (ASGCT).

Dr. George Stamatoyannopoulos, PhD, was a Greek geneticist whose research focused on inherited blood disorders, particularly anemias. He was a founder of the American Society of Gene and Cell Therapy in 1996. Since his passing in 2018, ASGCT has established a memorial lecture given in his honor during its annual meeting each year.

This year, the memorial lecture was introduced by Dr. Stephen Russell, PhD, the current president of ASGCT. During his opening remarks, Russell detailed the particular significance of this year's chosen speaker in the close ties to Stamatoyannopoulos' own passions. Like Stamatoyannopoulos, Sadelain has dedicated his career to developing therapies for hemoglobinopathies, specifically beta-thalassemia and sickle cell disease, Russell said.

Sadelin, who is a physician-scientist at Memorial Sloan Kettering (MSK) Cancer Center, is a pioneer in the field of chimeric antigen receptor (CAR) T-cell therapies. He was the founding director of the Center for Cell Engineering at MSK, and head of the gene transfer and gene expression lab at MSK. Sadelain also served as president of ASGCT from 2014 to 2015.

Russell explained that Sadelain became interested in generating retroviral vectors that express beta-globin with precise control over expression, a challenging project in the 1990s when tools were limited.

"If you have the intelligence, tenacity, and determination to achieve something in this field then you can achieve it," Russell commented during the session. "And I think that is what Michel has taught me."

Tenacity required to develop gene therapies

During his lecture, titled "Gene therapy through the lens of the beta-globin gene," Sadelin detailed his journey to developing lentiviral vectors that would allow for enough beta-globin expression to treat a mouse with beta-thalassemia. He explained that this took 11 years to accomplish, including five years in a postdoctoral position and six additional years running his own lab at MSK.

Together, beta-thalassemia and sickle cell disease represent the most common monogenic blood disorders. The related genetic disorders affect the beta-globin gene in human chromosome 11, which is relatively small with only three exons and two introns.

Beta-thalassemia is caused by a variety of different mutations (449 identified), mostly affecting the globin gene itself. These mutations result in insufficient expression of the beta chain of hemoglobin. The resulting imbalance between the alpha chain and beta chain production impairs erythropoiesis (red blood cell development).

The deficiency leads to hemolysis (destruction of red blood cells) and causes anemia (low levels of red blood cells). Importantly, Sadelain explained that beta-thalassemia can be treated with transfusions. The transfusions are lifesaving but are lifelong with serious risk of iron overload.

Alternatively, sickle cell disease only has one mutation in codon 6 of the globin gene that changes the properties of the hemoglobin protein. This results in normal hemoglobin production but with beta chains that bear a mutation, leading to the polymerization of hemoglobin in red blood cells. This causes the cells to become rigid and acquire the sickle shape, leading to vaso-occlusion, pain, strokes, and even death.

More recently, research has demonstrated that allogeneic bone marrow transplantation, in which hematopoietic stem cells (HSCs) from human leukocyte antigen (HLA)-matched sibling donors are given once, can be an effective treatment for these hemoglobinopathies. However, most patients do not have a match, and therefore require globin gene transfer from their own HSCs.

This provides a fundamental genetic rationale for treating the diseases, Sadelin said. He went on to explain foundational work in the late 1990s and early 2000s which was unsuccessful but offered a guide for what it would take to make a vector that encodes a functional globin gene.

One of the primary problems in the early days was that vectors did not produce enough of the globin gene. In beta-thalassemia and sickle cell disease, the therapeutic beta-globin gene needs to be produced at elevated (yet safe) levels in order to fill up all red blood cells with the correct anemia.

With various iterations of lentiviral vectors, Sadelain and his team were able to achieve correction of anemia in mice that lack beta-globin genes. He noted that the specific combination of promoters and locus control regions enabled the gene correction. However, while several versions of the vector were able to correct the gene, they also produced various copy numbers, which caused researchers to pause and assess the safety of the potential therapy.

A main safety concern for lentiviral gene therapies is clonal expansion, where individual genetic changes to cells lead to cancer development. This is the reason why beta-thalassemia and sickle cell disease gene therapy vectors must be erythrocyte-specific, Sadelain said. If the vectors are not specific to red blood cells, and they "integrate" in other tissues, then the chances of clonal expansion are greater. Therefore, monitoring to ensure that lentiviral vectors do not lead to clonal expansion of cancerous cell types is critical for assessing the safety of these gene therapy products moving forward.

One strategy that can help prevent clonal expansion is the inclusion of chromatin insulators, according to Sadelain, which could block interactions at the site of integration at neighboring elements of nonerythrocytes and prevent leaky expression of progenitor myeloid cell types.

Looking toward the future of HSC therapies, technologies like CRISPR-Cas9 or transcription activator-like effector nuclease (TALEN) can be used for beta-globin gene addition or hemoglobin gene correction. Sadelain noted that these synthetic biology approaches can be used in combination with cell therapy to enhance gene transfer levels and improve vectors.

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