MSGID: 1:317/3 64a3a08a   
   PID: hpt/lnx 1.9.0-cur 2019-01-08   
   TID: hpt/lnx 1.9.0-cur 2019-01-08   
    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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   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."   
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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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