Fred Hutchinson Cancer Center

University of Washington

The University of Arizona

Neutron Therapies Inc.

Fred Hutchinson Cancer Research Center

Mark T Groudine

Leland H Hartwell

Sharon E Plon

Ted A Weinert

Marion Frederick Hawthorne

Ryo Takeuchi

Barry L Stoddard

Harold M Weintraub, Deceased

Elliot M Epner

Ellen S Dieken

Norbert Kraut

C M Chen

Michael A Bender

R E Fournier

Provided are compositions and methods for the treatment of hemoglobinopathies such as thalassemias and sickle cell disease. Compositions and methods include one or more endonuclease(s) or endonuclease fusion protein(s), including one or more homing endonuclease(s) and/or homing endonuclease fusion protein(s) and/or CRISPR endonuclease(s) and/or CRISPR endonuclease fusion protein(s): (a) to disrupt a Bcl11a coding region; (b) to disrupt a Bcl11a gene regulatory region; (c) to modify an adult human &#x3b2;-globin locus; (d) to disrupt a HbF silencing DNA regulatory element or pathway, such as a Bcl11a-regulated HbF silencing region; (e) to mutate one or more &#x3b3;-globin gene promoter(s) to achieve increased expression of a &#x3b3;-globin gene; (f) to mutate one or more &#x3b4;-globin gene promoter(s) to achieve increased expression of a &#x3b4;-globin gene; and/or (g) to correct one or more &#x3b2;-globin gene mutation(s).

Compositions and methods for the treatment of hemoglobinopathies

Boron containing compounds are targeted to hormonally responsive cells by contacting the cells with a conjugate of a boron containing moiety conjugated to a ligand having binding specificity for an intracellular receptor of the cell. The conjugates are useful for imaging or killing hormonally responsive cells, such as hormonally responsive tumor cells. For target cell killing, the cells are contacted with a .sup.10 B containing moiety conjugated to a ligand having binding specificity for an intracellular hormone receptor of the cells. The .sup.10 B containing moiety becomes associated with the hormone receptor of the cells, which may then be irradiated with neutrons to kill the cells by boron neutron capture. For target cell imaging, the cells are contacted with a .sup.11 B containing moiety conjugated to a ligand having binding specificity for an intracellular hormone receptor of the cells, and the boron that becomes associated with the hormone receptor of the cells may then be imaged using magnetic resonance imaging techniques.

Boron-containing hormone analogs and methods of their use in imaging or

A novel gene, Inhibitor of MyoD Family (I-mf), is provided which encodes novel proteins, I-mfa, I-mfb and I-mfc, involved in regulation of myogenesis during vertebrate development. I-mf is highly expressed in the sclerotome of developing vertebrates and is postulated to play an important role in patterning of the somite and determination sclerotomal cell fate. A unique, C-terminal interactional domain of the I-mf protein mediates physical interactions between I-mfa and members of the MyoD family of transcriptional activators and functions to inhibit transactivation of muscle specific genes by MyoD family members, thereby repressing myogenesis. Further characterization of I-mf activity shows that I-mf associates with MyoD family member proteins and retains them in the cytoplasm by masking their nuclear localization signals. Based on the I-mf genes and proteins disclosed herein, a variety methods and compositions are provided for screening, isolating, and characterizing endogenous and exogenous factors, drugs and therapeutic agents useful to evaluate and/or control myogenesis normal and abnormal development and cell differentiation.

Polynucleotide sequences encoding proteins involved in myogenesis

Human checkpoint huCDC34, huRAD9.sub.compA, and huRAD9.sub.compB cDNAs shown in FIGS. 1, 2, and 3. A method for isolating a human checkpoint cDNA that is capable of restoring growth at a restrictive temperature in a yeast test cell, wherein the yeast test cell comprises a genome having a first gene that forms a DNA strand break at a restrictive temperature and a second gene that fails to induce a cell cycle arrest in response to the DNA strand break, whereby the growth of the yeast test cell is inhibited at the restrictive temperature, the method comprising the steps of: obtaining a human cDNA library comprising a plurality of human cDNA clones; inserting the human cDNA clones individually into plasmid vectors comprising a selectable marker gene; transforming a culture of the yeast test cells with the plasmid vectors from the preceding step; selecting for yeast test cells transformed with the selectable marker gene; growing the selected transformants at the restrictive temperature and isolating a candidate transformant capable of growing at the restrictive temperature; and identifying the human cDNA carried by the candidate transformant as a human checkpoint cDNA by sequencing the human cDNA carried by the candidate transformant and determining that the human cDNA is less than 50% homologous with both the first gene and the second gene. Also yeast checkpoint RAD17, RAD24, MEC1, MEC2, and MEC3 cDNAs shown in FIGS. 4-8.

Cell cycle checkpoint genes

Human checkpoint huCDC34, huRAD9.sub.compA, and huRAD9.sub.compB cDNAs shown in SEQ ID Nos:7-9. A method for isolating a human checkpoint cDNA that is capable of restoring growth at a restrictive temperature in a yeast test cell, wherein the yeast test cell comprises a genome having a first gene that forms a DNA strand break at a restrictive temperature and a second gene that fails to induce a cell cycle arrest in response to the DNA strand break, whereby the growth of the yeast test cell is inhibited at the restrictive temperature, the method comprising the steps of: obtaining a human cDNA library comprising a plurality of human cDNA clones; inserting the human cDNA clones individually into plasmid vectors comprising a selectable marker gene; transforming a culture of the yeast test cells with the plasmid vectors from the preceding step; selecting for yeast test cells transformed with the selectable marker gene; growing the selected transformants at the restrictive temperature and isolating a candidate transformant capable of growing at the restrictive temperature; and identifying the human cDNA carried by the candidate transformant as a human checkpoint cDNA by sequencing the human cDNA carried by the candidate transformant and determining that the human cDNA is less than 50% homologous with both the first gene and the second gene. Also yeast checkpoint RAD17, RAD24, MEC1, MEC2, and MEC3 cDNAs shown in SEQ ID Nos:10-19.

An avian/mammalian microcell hybrid immortalized pre B cell line containing a mammalian chromosome that carries a selectable marker has been produced. The avian/mammalian microcell hybrid immortalized pre B cell line can be used for introducing predetermined point mutations into specific mammalian chromosomal loci via high frequency homologous recombination.

Recombination-proficient avian/mammalian microcell hybrids

Growing community of inventors

Seattle, WA, United States of America

Mark T Groudine