TGR - Sickle Cell
File Created On:09/22/2002 08:51:35 PM
Subject:Sickle Cell
(The following material is presented in Ascending Date Order)
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Researcher Name shown is my estimate of Principal Researcher
DB Key:1996-SC-A
Doc:Abstract
Vector:RDO RNA-DNA chimeric oligonucleotide
Experiment:Failed or Not Applicable
Of Note:As few as 10% normal hematopoietic cells
9/1/1996-----Kmiec EB-----Thomas Jefferson University
Title:Correction of the mutation responsible for sickle cell anemia by an RNA-DNA oligonucleotide
Link:http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=8703073&form=6&db=m&Dopt=r
Disease Information:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10791557&dopt=Abstract
Snippet:
A chimeric oligonucleotide composed of DNA and modified RNA residues was used to direct correction of the mutation in the hemoglobin betaS allele. After introduction of the chimeric molecule into lymphoblastoid cells homozygous for the betaS mutation, there was a detectable level of gene conversion of the mutant allele to the normal sequence. The efficient and specific conversion directed by chimeric molecules may hold promise as a therapeutic method for the treatment of genetic diseases
DB Key:1997-SC-A
Doc:Abstract
Vector:RDO RNA-DNA chimeric oligonucleotide
Experiment:Failed or Not Applicable
Of Note:5-11% conversion rate was measured
11/1/1997-----Kmiec EB-----Thomas Jefferson University
Title:Targeted gene conversion in a mammalian CD34+-enriched cell population using a chimeric RNA/DNA oligonucleotide
Link:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9428613&dopt=Abstract
Disease Information:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10791557&dopt=Abstract
Snippet:
Gene conversion of genetically inherited point mutations is a fundamental methodology for treating a variety of diseases. We tested the feasibility of a new approach using an RNA/DNA chimeric oligonucleotide. The beta-globin gene was targeted at the point mutation causing sickle cell anemia. The chimera is designed to convert an A residue to a T after creating a mismatched basepair. In a CD34+-enriched population of normal cells a 5-11% conversion rate was measured using restriction enzyme polymorphism and direct DNA sequence analyses. The closely related delta-globin gene sequence appeared unchanged despite successful conversion at the beta-globin locus.
Gene conversion of genetically inherited point mutations is a fundamental methodology for treating a variety of diseases. We tested the feasibility of a new approach using an RNA/DNA chimeric oligonucleotide. The beta-globin gene was targeted at the point mutation causing sickle cell anemia. The chimera is designed to convert an A residue to a T after creating a mismatched basepair. In a CD34+-enriched population of normal cells a 5-11% conversion rate was measured using restriction enzyme polymorphism and direct DNA sequence analyses. The closely related delta-globin gene sequence appeared unchanged despite successful conversion at the beta-globin locus.
DB Key:1999-SC-A
Doc:Abstract
Vector:NA Not Applicable
Experiment:Failed or Not Applicable
Of Note:The yeast system produces a recombinant sickle Hb that is identical by about a dozen biochemical and physiological criteria with the natural sickle Hb purified from the red cells of sickle-cell anaemia patients. Most importantly, the gelling concentration of this recombinant sickle Hb is the same as that of the HbS purified from human sickle red cells.
4/1/1999-----Li,Xianfeng-----Northeastern University
Title:Mutational analysis of sickle haemoglobin (Hb) gelation
Link:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10075913&dopt=Abstract
2nd Link:http://bab.portlandpress.com/bab/029/bab0290165.htm
Disease Information:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10791557&dopt=Abstract
Snippet:
We have recently reported the expression of human sickle Hb (HbS) in the yeast Saccharomyces cerevisiae that carries a plasmid containing the human - and b-globin cDNA sequences; N-terminal nascent protein processing is correct and a soluble correctly folded Hb tetramer is produced. The yeast system produces a recombinant sickle Hb that is identical by about a dozen biochemical and physiological criteria with the natural sickle Hb purified from the red cells of sickle-cell anaemia patients. Most importantly, the gelling concentration of this recombinant sickle Hb is the same as that of the HbS purified from human sickle red cells. The misfolding of Hb reported for the Escherichia coli-expressed protein is not apparent for Hb expressed in yeast by any of the criteria that we have used for characterization
DB Key:1999-SC-B
Doc:American Scientist
Vector:RDO RNA-DNA chimeric oligonucleotide
Experiment:Failed or Not Applicable
Of Note:For example, sickle cell anemia is the result of a single nucleotide substitution, a single letter misspelled, in the gene encoding the b-globin strand of hemoglobin
5/1/1999-----Kmiec EB-----Thomas Jefferson University
Title:Targeted Gene Repair
Link:http://www.americanscientist.org/articles/99articles/kmiecrepair.html
Disease Information:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10791557&dopt=Abstract
Snippet:
Our work builds on earlier studies from Fred Sherman’s laboratory at the University of Rochester, who used a similar technique to change a single nucleotide. But the oligomers used by the Sherman group were unstable, and the team never extended its work. It turns out that mammalian cells contain enzymes that either degrade the ends of DNA molecules, or link them in long arrays called concatamers, which essentially destroys the integrity of the oligomers. We discovered that we could increase the stability of an oligomer by attaching segments of RNA to each of its ends. Like DNA, RNA is also composed of strings of nucleotides and therefore can bind to DNA in the same complementary manner as can another strand of DNA. (RNA contains no thymine. Instead, the uracil in RNA pairs with the adenine in DNA.)
DB Key:2000-SC-A
Doc:Abstract
Vector:TFO Triple helix-forming oligonucleotide
Experiment:Failed or Not Applicable
Of Note:We have explored the possibility of activating the gamma-globin gene expression by triplex-forming oligonucleotide (TFO)-directed targeted mutagenesis.
1/1/2000-----Glazer PM-----University of South Alabama College of Medicine
Title:Activation of human gamma-globin gene expression via triplex-forming oligonucleotide (TFO)-directed mutations in the gamma-globin gene 5' flanking region
Link:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10721715&dopt=Abstract
Disease Information:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10791557&dopt=Abstract
Snippet:
Human beta-globin disorders, such as sickle cell anemia and beta-thalassemia, are relatively common genetic diseases cause by mutations in the beta-globin gene. Increasing gamma-globin gene expression has been found to greatly reduce the disease symptom. However, the gamma-globin gene is developmentally regulated and normally expressed at high levels only during the fetal stage of human development. We have explored the possibility of activating the gamma-globin gene expression by triplex-forming oligonucleotide (TFO)-directed targeted mutagenesis. Using a psoralen-conjugated TFO designed to bind to a site overlapping with an Oct-1 binding site at the -280 region of the gamma-globin gene, targeted mutagenesis of the Oct-1 binding site has been achieved by transfecting the in-vitro-formed plasmid-oligo complex into human normal fibroblast (NF) cells. The mutation frequency at the target site was estimated to be 20% by direct DNA sequencing analysis. In-vitro protein binding assays indicated that these mutations reduced Oct-1 binding to the target site. In-vivo gene expression assays demonstrated activation of gamma-globin gene expression from these mutations in mouse erythroleukemia (MEL) cells. The levels of the gamma-globin gene expression increased by as much as fourfold in mutants with single base changes. These results suggest that the -280 region of the Agamma-globin gene negatively regulates the gamma-globin gene expression, and mutations at the Oct-1 binding site can lead to activation of the gamma-globin gene and generate the hereditary persistence of fetal hemoglobin (HPFH) condition. This study may provide a novel approach for gene therapy of sickle cell disease. The data may also have implications in gene therapy for other diseases including genetic diseases and cancers by introduc
DB Key:2001-SC-A
Doc:Abstract
Vector:RDO RNA-DNA chimeric oligonucleotide
Experiment:Failed or Not Applicable
Of Note:Saccharomyces Cerevisae
5/1/2001-----Kmiec EB-----University of Delaware
Title:In vitro and in vivo nucleotide exchange directed by chimeric RNA/DNA oligonucleotides in Saccharomyces cerevisae
Link:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11401693&dopt=Abstract
Disease Information:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10791557&dopt=Abstract
Snippet:
To this end, we have identified genes that control targeted repair, using the genetically tractable organism, Saccharomyces cerevisae and a bank of yeast mutants
DB Key:2001-SC-O
Doc:Abstract
Vector:RDO RNA-DNA chimeric oligonucleotide
Experiment:Failed or Not Applicable
Of Note:Here, the utility of this vector is expanded into Saccharomyces cerevisiae
10/15/2001-----Kmiec EB-----University of Delaware
Title:In vivo gene repair of point and frameshift mutations directed by chimeric RNA/DNA oligonucleotides and modified single-stranded oligonucleotides
Link:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11600713&dopt=Abstract
2nd Link:http://nar.oupjournals.org/cgi/content/abstract/29/20/4238?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&author1=Kmiec+EB&searchid=1032104998174_674&stored_search=&FIRSTINDEX=0&sortspec=Score+desc+PUBDATE_SORTDATE+desc&journalcode=nar
Snippet:
Among the most promising vectors is chimeric oligonucleotide (CO), a double-stranded, RNA-DNA hybrid molecule folded into a double hairpin conformation: by using the cell's DNA repair machinery, the CO directs nucleotide exchange as episomal and chromosomal DNA. Systematic dissection of the CO revealed that the region of contiguous DNA bases was the active component in the repair process, especially when the single-stranded ends were protected against nuclease attack. Here, the utility of this vector is expanded into Saccharomyces cerevisiae. An episome containing a mutated fusion gene encoding hygromycin resistance and eGFP expression was used as the target for repair
DB Key:2002-SC-A
Doc:Abstract
Vector:RDO RNA-DNA chimeric oligonucleotide
Experiment:Failed or Not Applicable
Of Note:Human Cell Success
1/1/2002-----Kmiec EB-----University of Texas Medical Branch
Title:Targeted beta-globin gene conversion in human hematopoietic CD34(+ )and Lin(-)CD38(-)cells.
Link:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11857070&dopt=Abstract
Disease Information:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10791557&dopt=Abstract
Snippet:
Chimeric oligonucleotides have been used successfully to correct point and frameshift mutations in several cell types, as well as in animal and plant models. However, their application to primitive human blood cells has been limited. In this investigation, chimeric oligonucleotides designed to direct a site-specific nucleotide exchange in the human beta-globin gene (an A to T substitution within codon 6) were introduced into normal human CD34(+) and Lin(-)CD38(-) cells via microinjection. This A to T nucleotide exchange introduces the single site mutation responsible for sickle cell anemia. In 23% of experimental samples, gene conversion was detected in the progeny of microinjected CD34(+) and Lin(-)CD38(-) cells that were cultured for at least 4 weeks. In addition, gene conversion was detected in the erythroid progeny of Lin(-)CD38(-) cells at the mRNA level. Conversion rates as high as 10-15% in 11% (five of 44) of experimental samples were confirmed by allele-specific PCR and sequence analysis of genomic DNA from the progeny of microinjected Lin(-)CD38(-) cells. Given that as few as 10% normal hematopoietic cells are sufficient to keep patients free of sickle cell disease, the level of conversion we have achieved in some samples may well be of therapeutic benefit in patients with sickle cell disease.
DB Key:2002-SC-C
Doc:
Vector:
Experiment:Failed or Not Applicable
Of Note:A turning point came from Michel Sadelain's lab at Memorial Sloan-Kettering Cancer Center in New York. In 2000,2 Sadelain detailed the first successful use of an HIV-based lentiviral vector for the long-term correction of the blood disorder b-thalassemia, in which the body makes too little hemoglobin. Murine models treated in Sadelain's lab continue to show correction of the disorder, he notes.
In 2001, LeBoulch's team used an HIV-based vector to transplant healthy stem cells carrying a specially manipulated bA-globin gene called gemisch into murine models. The gene therapy resulted in almost 100% expression of functioning red blood cells with anti-sickling globin and corrected all SCD symptoms. The mice have maintained this rate for more than a year, Leboulch reports.
3/18/2002-----Sadelain,Michel-----Memorial Sloan-Kettering Cancer Center
Title:Murine Gene Therapy Corrects Symptoms of Sickle Cell Disease
Link:http://www.the-scientist.com/yr2002/mar/research3_020318.html
Snippet:
Caused by a simple gene mutation that misshapes red blood cells and renders them ineffective, sickle cell disease (SCD) seemed to provide scientists with a straightforward target for gene therapy. But since the first SCD gene therapy experiment in 1979, researchers continue to struggle. It wasn't until late 2001, in a paper listed in the Faculty of 1000's Top 10 Genomics list for Feb. 15, that scientists corrected the murine symptoms of this chronic, inherited, and often painful disorder.1
The successful pathway proved far more complex and challenging than initially imagined, says lead investigator gene therapist Philippe Leboulch, of the Massachusetts Institute of Technology and Harvard University. "Everybody thought it would be the first genetic disorder cured by gene therapy, that it would be simple, but it turned out to be completely different. It was a real challenge," says Leboulch, who has worked with SCD for more than 10 years.
Two key factors made SCD a promising candidate for gene therapy research. First, the sickle cell mutation is a single point alteration in the human bA-globin gene, which causes the formation of an abnormal hemoglobin. Second, scientists could isolate bone marrow stem cells relatively easily and introduce potential anti-sickling genes ex vivo. But success required overcoming difficult problems such as delivering a normal gene to the body, manipulating the gene for long-term expression and a high level of effectiveness, and creating mouse models.
A turning point came from Michel Sadelain's lab at Memorial Sloan-Kettering Cancer Center in New York. In 2000,2 Sadelain detailed the first successful use of an HIV-based lentiviral vector for the long-term correction of the blood disorder b-thalassemia, in which the body makes too little hemoglobin. Murine mode
DB Key:2002-SFHR-B6
Doc:Abstract
Vector:SFHR Small Fragment DNA
Experiment:Failed or Not Applicable
Of Note:The potential for one gene targeting technique, small fragment homologous replacement (SFHR) to the gene therapy treatment of sickle cell disease (SCD) is presented
6/1/2002-----Goncz KK-----University of Vermont
Title:Application of SFHR to gene therapy of monogenic disorders
Link:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12032689&dopt=Abstract
Disease Information:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10791557&dopt=Abstract
Snippet:
The potential for one gene targeting technique, small fragment homologous replacement (SFHR) to the gene therapy treatment of sickle cell disease (SCD) is presented
DB Key:2002-SC-E
Doc:Biography
Vector:RDO RNA-DNA chimeric oligonucleotide
Experiment:Failed or Not Applicable
Of Note:In my laboratory, we use the yeast Saccharomyces cerevisiae to study the genetics and biochemistry of recombination. Malfunctions of this important cellular process can cause loss of heterozygosity, a mechanism known to lead to tumorigenesis for some cancers. For example, one of the yeast genes that we isolated during our recombination studies has homologs implicated in two human disorders, Bloom and Werner syndromes, both of which cause an increased risk of cancer
7/1/2002-----Rothstein,Rodney-----Columbia University
Title:Biography
Link:http://icg.cpmc.columbia.edu/faculty_Rothstein.htm
Disease Information:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10791557&dopt=Abstract
Snippet:
By searching for mutations that increase recombination, we have successfully identified new genes and functions involved in suppressing recombination between naturally occurring repetitive elements dispersed throughout the genome. The analysis of these hyper-recombination mutations has resulted in the identification of: TOP3, a novel DNA topoisomerase; SGS1, a helicase that defines the gene family for Bloom and Werner syndrome; POL12, a DNA polymerase alpha-associated factor and RFA1, the yeast single-stranded DNA binding protein. Each gene is evolutionarily conserved from bacteria to man, illustrating the importance of their function in all organisms
DB Key:2002-SC-F
Doc:Abstract
Vector:RDO RNA-DNA chimeric oligonucleotide
Experiment:Successful
Of Note:Genetic analyses reveal that expression levels of the recombination/repair genes RAD51, RAD52 and RAD54 can affect the frequency of gene repair. Overexpression of RAD51 enhances the frequency 4-fold for correction of an episomal target and 5-fold for correction of a chromosomal target; overexpression of RAD54 is also effective in stimulating gene repair, to the same extent as RAD51 in the chromosomal target. In sharp contrast, RAD52 gene expression serves to reduce gene repair activity in rescue experiments and in experiments where RAD52 is overexpressed in a wild-type strain.
7/15/2002-----Kmiec EB-----University of Delaware
Title:Rad51p and Rad54p, but not Rad52p, elevate gene repair in Saccharomyces cerevisiae directed by modified single-stranded oligonucleotide vectors
Link:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12087156&dopt=Abstract
2nd Link:http://nar.oupjournals.org/cgi/content/abstract/30/13/2742?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&author1=Kmiec+EB&searchid=1032104998174_674&stored_search=&FIRSTINDEX=0&sortspec=Score+desc+PUBDATE_SORTDATE+desc&journalcode=nar
Disease Information:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10791557&dopt=Abstract
Snippet:
In sharp contrast, RAD52 gene expression serves to reduce gene repair activity in rescue experiments and in experiments where RAD52 is overexpressed in a wild-type strain. This may suggest an antagonist role for Rad52p. Consistent with this notion, the highest level of targeted repair occurs when the RAD51 gene is overexpressed in a strain of yeast deficient in RAD52 gene function
DB Key:2002-SC-J
Doc:Abstract
Vector:HR Homologous Recombination
Experiment:Failed or Not Applicable
Of Note:Saccharomyces cerevisiae Rad51, Rad54, and RPA proteins work in concert to make heteroduplex DNA joints during homologous recombination. With plasmid length DNA substrates, maximal DNA joint formation is observed with amounts of Rad51 substantially below what is needed to saturate the initiating ssDNA template, and, relative to Rad51, Rad54 is needed in only catalytic quantities
9/10/2002-----Van Komen S-----UT Health Science Center at San Antonio
Title:Functional crosstalk among Rad51, Rad54, and RPA in Heteroduplex DNA joint formation
Link:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12226081&dopt=Abstract
Snippet:
We also demonstrate that Rad54 helps overcome various reaction constraints in DNA joint formation. These results thus shed light on the function of Rad54 in the Rad51-mediated homologous DNA pairing reaction and also reveal a novel role of RPA in the presynaptic stage of this reaction. |
