Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-07-27
2002-09-17
Nguyen, Dave T. (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S325000, C435S370000, C435S375000, C435S455000, C536S023100, C536S023500
Reexamination Certificate
active
06451558
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to nucleic acid and amino acid sequences of three novel genes involved in the control of hematopoiesis.
BACKGROUND OF THE INVENTION
Hematopoiesis (used interchangeably with hemopoiesis) is a process whereby multi-potent stem cells give rise to lineage-restricted progeny. Hemopoietic stem cells (HSCs) are the only cells in the hematopoietic system that produce other stem cells and give rise to the entire range of blood and immune system cells. In humans, CD34
+
Thy-1
+
Lin
−
cells from bone marrow and mobilized peripheral blood are highly enriched for HSCs (Murray et al.,
Blood Cells,
20:354-370 (1995a); Murray et al.,
Blood,
85:368-378 (1995b)). This cell population is capable of self-renewal and long term multilineage differentiation and has been successfully used for autologous transplantation (Gazitt et al.,
Blood,
86:381-389 (1995)). Since HSCs self-renew and are multi-potent, they are ideal candidates for gene therapy. Gene therapy is a new treatment modality for a variety of genetic, neoplastic, or infectious diseases and has the potential to correct defects in all mature cells derived from HSC.
The molecular basis of hematopoiesis remains poorly understood. A greater understanding of the process whereby HSCs give rise to lineage-restricted progeny would facilitate the exploitation of HSCs for transplantation and gene therapy. To achieve this, the molecular pathways controlling hemopoietic cell growth and differentiation have been investigated. To this end, the present invention concerns the identification of candidate HSC regulatory genes and their impact on hematopoiesis.
In the present invention, a cDNA library has been built and characterized from human CD34
+
Thy-1
+
stem cells. Sequence analysis of the cDNA library revealed a high degree of novel proteins which may play a role in hematopoiesis. To enrich for candidate hemopoietic stem cell (HSC) regulatory genes, RNA expression profiling was performed and cDNAs whose expression was enriched in HSC were selected and compared to other differentiated blood cell types. Candidate genes were fully sequenced. The HSC-enriched genes which encode full length novel gene products were subcloned into a retroviral expression vector, which was used to overexpress the gene product in freshly isolated HSCs. Three novel HSC regulatory genes and the proteins which they encode were identified. Each of the novel cDNAs are enriched in HSCs compared to their differentiated progeny, and when overexpressed in HSCs blocks the differentiation of stem cells.
SUMMARY OF THE INVENTION
The present invention discloses three novel genes involved in HSC regulation, hereinafter referred to as SCM 26, SCM 3, and SCM 113.
In a first embodiment, the invention concerns isolated polynucleotide sequences encoding a polypeptide comprising an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO. 2; the amino acid sequence of SEQ ID NO. 4; the amino acid sequence of residues 1-239 of SEQ ID NO.4; the amino acid sequence of residues 240-543 of SEQ ID NO. 4; the amino acid sequence of SEQ ID NO. 6; and an amino acid sequence functionally equivalent to the above enumerated sequences.
In a second embodiment, the invention relates to an isolated DNA sequence comprising a nucleotide sequence selected from the group consisting of the polynucleotide sequence of SEQ ID NO 1; the polynucleotide sequence of SEQ ID NO. 3; the polynucleotide sequence of SEQ ID NO. 5; and a polynucleotide sequence at least 85% identical to a polynucleotide sequence as disclosed above. In one aspect, the isolated polynucleotide sequence may consist of the complement of the polynucleotide sequences of those listed above.
In a third embodiment, the invention concerns, an isolated polypeptide comprising a member selected from the group consisting of, a polypeptide comprising the amino acid sequence of SEQ ID NO. 2; a polypeptide comprising the amino acid residues 26-40 of SEQ ID NO. 2; a polypeptide comprising the amino acid residues 25-82 of SEQ ID NO. 2; a polypeptide comprising the amino acid residues 147-157 of SEQ ID NO. 2; a polypeptide comprising the amino acid residues 266-275 of SEQ ID NO. 2; a polypeptide comprising the amino acid sequence of SEQ ID NO. 4; a polypeptide comprising the amino acid residues 1-239 of SEQ ID NO. 4; a polypeptide comprising the amino acid residues 240-543 of SEQ ID NO. 4; a polypeptide comprising the amino acid sequence of SEQ ID NO. 6; or a polypeptide having at least 85% identity to said polypeptides disclosed above. In a preferred aspect, the isolated polypeptide comprises the amino acid sequence of SEQ ID Nos. 2, 4, 6, or a polypeptide having an amino acid sequence with 95% identity thereto. In another aspect, the invention relates to an isolated polypeptide comprising a member selected from the group consisting of the amino acid residues 26-40 of SEQ ID NO. 2; the amino acid residues 25-82 of SEQ ID NO. 2; the amino acid residues 147-157 of SEQ ID NO. 2; the amino acid residues 266-275 of SEQ ID NO. 2; and a polypeptide having at least 97% identity thereto. In yet a further aspect, the invention relates to a DNA sequence encoding one of the above enumerated polypeptides.
In a fourth embodiment, the invention concerns a vector which incorporates one of the claimed polynucleotide sequences of the invention. In a preferred aspect, the vector is a retroviral vector, adenoviral vector, or adeno-associated vector. In a further preferred aspect, a host cell is claimed which includes the vector. A preferred host cell is a hematopoietic cell, particularly a human CD34
+
cell.
In a fifth embodiment, the invention concerns a method of increasing the effective dose of hematopoietic stem cells in a mammalian subject, comprising obtaining a population of CD34
+
cells which includes a subpopulation of hematopoietic stem cells; introducing into the CD34
+
cells a polynucleotide sequence of the invention encoding a disclosed polypeptide of the invention; obtaining a subpopulation of genetically modified stem cells which overexpress said polypeptide; and administering said subpopulation of genetically modified cells to a subject wherein the effective dose of the hematopoietic stem cells is increased. In a further aspect, the invention includes the step of selecting hematopoietic stem cells either prior to or after genetic modification. In yet another aspect, the invention includes the step of culturing the population of hematopoietic CD34
+
cells either prior to or after genetic modification.
In a sixth embodiment the invention concerns, a method of increasing the effective dose of gene modified cells comprising obtaining a population of hematopoietic CD34
+
cells which includes a subpopulation of hematopoietic stem cells; introducing into the population of CD34
+
cells a polynucleotide sequence of the invention; introducing into the population of CD34
+
cells a second polynucleotide sequence wherein said second polynucleotide sequence encodes a therapeutic gene; obtaining genetically modified cells wherein said cells are capable of expressing the polynucleotide sequence of the invention and the therapeutic gene wherein the effective dose of the cells capable of expressing the therapeutic gene is increased compared to wild-type cells; and administering the genetically modified cells to a mammalian subject.
In an seventh embodiment, the invention concerns a method of blocking the differentiation of mammalian hematopoietic stem cells in vitro comprising the steps of; isolating CD34
+
cells from a source of hematopoietic cells; introducing a vector comprising the claimed polynucleotide sequences into the CD34
+
cells, whereby a population of said cells are genetically modified by said vector; culturing the modified CD34
+
cells in the presence of at least one cytokine in an amount sufficient to support growth of the modified cells; and selecting cells in which the polypeptide is over
Cooke Michael Paul
Holness Claire Louise
Sirenko Oksana Ivanivna
Golightly Douglas A.
Hoxie Thomas
Karny Geoffrey M.
Nguyen Dave T.
Nguyen Quang
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