Base editing shows potential superiority for curing sickle cell disease


Date:
July 3, 2023
Source:
St. Jude Children's Research Hospital
Summary:
Adenosine base editing restarted fetal hemoglobin expression in
cells from patients with sickle cell disease.


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FULL STORY ==========================================================================
Gene therapy that alters hemoglobin genes may be an answer to curing
sickle cell disease (SCD) and beta thalassemia. These two common life-threatening anemias afflict millions of individuals across the
globe. Scientists at St.

Jude Children's Research Hospital and the Broad Institute of MIT and
Harvard used a next-generation genome editing technology, adenosine base editing, to restart fetal hemoglobin expression in SCD patient cells. The approach raised the expression of fetal hemoglobin to higher, more stable,
and more uniform levels than other genome editing technologies that use CRISPR/Cas9 nuclease in human hematopoietic stem cells. The findings
were published today in Nature Genetics.

SCD and beta thalassemia are blood disorders affecting millions of people; mutations in the gene that encodes an adult version of the oxygen-carrying molecule hemoglobin cause these disorders. Restoring gene expression
of an alternative hemoglobin subunit active in a developing fetus
has previously shown therapeutic benefit in SCD and beta thalassemia
patients. The researchers wanted to find and optimize genomic technology
to edit the fetal hemoglobin gene. One alteration installed by adenosine
base editing was particularly potent for restoring fetal hemoglobin
expression in post-natal red blood cells.

"We showed base editors meaningfully increase fetal hemoglobin
levels," said lead corresponding author Jonathan Yen, Ph.D., St. Jude Therapeutic Genome Engineering group director. "Now, my Therapeutic
Genome Engineering team is already hard at work, starting to optimize
base editing to move this technology to the clinic." Hemoglobin holds
the key Adult hemoglobin, expressed primarily after birth, contains four protein subunits -- two beta-globin and two alpha-globin. Mutations in
the beta-globin gene cause sickle cell disease and beta-thalassemia. But
humans have another hemoglobin subunit gene (gamma-globin), which is
expressed during fetal development instead of beta-globin. Gamma-globin combines with alpha-globin to form fetal hemoglobin. Normally around
birth, gamma-globin expression is turned off, and beta-globin is turned
on, switching from fetal to adult hemoglobin.

Genome editing technologies can introduce mutations that turn the
gamma-globin gene back on, thereby increasing fetal hemoglobin
production, which can effectively substitute for defective adult
hemoglobin production.

"We used a based editor to create a new TAL1 transcription factor binding
site that causes particularly strong induction of fetal hemoglobin,"
Yen said.

"Creating a new transcription factor binding site requires a precise base
pair change -- something that can't be done using CRISPR-Cas9 without generating unwanted byproducts and other potential consequences from double-stranded breaks." "The gamma-globin [fetal hemoglobin] gene is a
good target for base editing because there are very precise mutations that
can reactivate its expression to induce expression after birth, which may provide a powerful 'one-size-fits-all' treatment for all mutations that
cause SCD and beta-thalassemia," said co- corresponding author Mitchell
Weiss, M.D., Ph.D., St. Jude Department of Hematology chair.

Thus, scientists want to restore fetal hemoglobin expression because
it is a more universal treatment for major hemoglobin disorders than
correcting the SCD mutation or hundreds of mutations that cause beta thalassemia. Increasing fetal hemoglobin expression has the potential
to therapeutically benefit most patients with SCD or beta thalassemia, regardless of their causative mutations.

Researchers have previously shown proof-of-principle with multiple genome editing approaches, but this study is the first to systematically compare
these different strategies' efficacy.

"We looked closely at the individual DNA sequence outcomes of nucleases
and base editors used to make therapeutic edits of fetal hemoglobin
genes. Since nucleases often generate complex, uncontrolled mixtures
of many different DNA sequence outcomes, we characterized how each nuclease-edited sequence affects fetal hemoglobin expression. Then we did
the same for base editing outcomes, which were much more homogeneous,"said co-corresponding author David Liu, Ph.D., Richard Merkin, Professor
at Broad Institute of MIT and Harvard, whose lab invented base editing
in 2016.

The study discovered that using base editing at the most potent site
in the gamma-globin promoter achieved 2- to 4-fold greater HbF levels
than Cas9 editing. They further demonstrated that these base edits could
be retained in engrafting blood stem cells from healthy donors and SCD
patients by putting them into immunocompromised mice.

Addressing safety concerns "Ultimately, we showed that not all genetic approaches are equal," Yen said.

"Base editors may be able to create more potent and precise edits than
other technologies. But we must do more safety testing and optimization."
When compared for safety, base editing caused fewer genotoxic events,
such as p53 activation and large deletions. Base editing was much
more consistent in its edits and products -- a highly desirable safety
property for a clinical therapy. In contrast to conventional Cas9, which generates uncontrolled mixtures of insertion and deletion mutations
termed "indels," base editing generates precise nucleotide changes with
few undesired byproducts.

"In our comparison, we found unanticipated problems with conventional
Cas9 nucleases," Weiss said. "We were somewhat surprised that not every
Cas9 insertion or deletion raised fetal hemoglobin to the same extent, indicating the potential for heterogeneous biological outcomes with that technology." The group found that individual red blood cells derived
from hematopoietic stem cells treated with the same Cas9 produce a
more variable amount of fetal hemoglobin compared to cells treated
with base editing. Thus, base editing produced more potent, reliable,
and consistent outcomes, which are desirable therapeutic properties.

Though base editing performed well, researchers have yet to determine
its safety in patients. Notably, base editing may have some risks
not presented by Cas9; for example, some early base editors can cause
undesired changes in genomic DNA or RNA at off-target sites. The group
showed that these changes are relatively small and not predicted to be
harmful, but deeper studies are warranted to evaluate these risks fully.

The future of gene editing therapeutics Throughout the study, the
scientists directly compared the performance of Cas9 nucleases at
two different target sites that induce fetal hemoglobin production in
different ways and base editing. Base editing uses a distinct editing
mechanism that directly converts one DNA base pair to another, rather
than cutting the DNA double helix into two pieces.

The Cas9 nuclease approaches create mixtures of deletions and insertions
that impair the expression or activity of BCL11A, a well-known
gamma-globin gene repressor. In contrast, base editing creates a novel transcription factor binding motif within the gamma-globin promoter. The
Cas9 nuclease approaches and a different base editing approach are being
tested through clinical trials.

St. Jude is participating in some of these studies.

"It is very important to test and compare different genome editing
approaches for treating SCD and beta-thalassemia because the best ones
are not known," said Weiss.

John Tisdale, M.D., a study co-author and the Cellular and Molecular Therapeutics Branch chief at the National Heart, Lung, and Blood
Institute, agreed. "The science of gene editing is moving quickly, and
we are now able to envision multiple different strategies for combating
sickle cell disease," Tisdale said. "These findings bring us a step
closer to our goal of broadly available cures."
* RELATED_TOPICS
o Health_&_Medicine
# Sickle_Cell_Anemia # Anemia # Birth_Defects #
Genes # Gene_Therapy # Human_Biology # Stem_Cells #
Personalized_Medicine
* RELATED_TERMS
o Sickle-cell_disease o Natural_killer_cell o Stem_cell
o Somatic_cell o Cell_(biology) o Embryonic_stem_cell o
Chemotherapy o Axon

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========================================================================== Journal Reference:
1. Thiyagaraj Mayuranathan, Gregory A. Newby, Ruopeng Feng, Yu Yao,
Kalin D.

Mayberry, Cicera R. Lazzarotto, Yichao Li, Rachel M. Levine, Nikitha
Nimmagadda, Erin Dempsey, Guolian Kang, Shaina N. Porter, Phillip A.

Doerfler, Jingjing Zhang, Yoonjeong Jang, Jingjing Chen, Henry
W. Bell, Merlin Crossley, Senthil Velan Bhoopalan, Akshay Sharma,
John F. Tisdale, Shondra M. Pruett-Miller, Yong Cheng, Shengdar
Q. Tsai, David R. Liu, Mitchell J. Weiss, Jonathan S. Yen. Potent
and uniform fetal hemoglobin induction via base editing. Nature
Genetics, 2023; DOI: 10.1038/s41588- 023-01434-7 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2023/07/230703133055.htm

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