1. A DNA cassette for producing CD34 positive hematopoietic stem cells transfected with one or more copies of globin gene, one or more suicide genes including HSV-tk and Cytosine deaminase (but not exclusively these 2 suicide genes) expressed either individually or in combination such that both sequences are flanking the two sides of viral 2A peptide or internal ribosomal entry (IRES) sequence.

2. The DNA cassette for producing CD34 positive hematopoietic stem cells transfected with one or more copies of globin gene, as claimed in claim 1 wherein, the said therapeutic genes are expressed under any known conventional promoter, including but not exclusively an erythroid specific promoter like KLF-1 promoter.

3. The DNA cassette for producing CD34 positive hematopoietic stem cells transfected with one or more copies of globin gene as claimed in claim 1 wherein, the said therapeutic genes are expressed along with luciferase gene for easy monitoring of the transgenic cells and or along with erythropoietin gene to increase the number of red blood cells.

4. The DNA cassette for producing CD34 positive hematopoietic stem cells transfected with one or more copies of globin gene as claimed in claim 1 wherein, the said carrier cells utilized for expressing the said DNA cassette to form the normal red blood cells (RBCs), and thus treat and cure the hemoglobin related diseases in patients of thalassemia, sickle cell disease and other genetic diseases.

5. The DNA cassette for producing CD34 positive hematopoietic stem cells transfected with one or more copies of globin gene as claimed in claim 1, wherein the said DNA cassette allows constitutive or inducible expression of the said therapeutic genes within the selected carrier cells for producing normal red blood cells.

6. The DNA cassette for producing CD34 positive hematopoietic stem cells transfected with one or more copies of globin gene as claimed in claim 1 wherein, the said the DNA cassette is tagged with the appropriate selection marker like Bleomycin/ Zeocin, Green fluorescent protein or any small DNA tag (His/FLAG tag etc. to confirm the functional expression of the said DNA cassette visually.

7. The DNA cassette for producing CD34 positive hematopoietic stem cells transfected with one or more copies of globin gene as claimed in claim 1 wherein, the therapeutic assembly can be contained within the inverted terminal repeats of transposons so as to provide permanent expression in cells without use of viral mode of transfection and use of DNA minicircles so that the patients can be treated with a simple intravenous injection or infusion provided to the patient in one or multiple settings (single injection or repeated injections) till complete symptomatic relief is obtained from the disease.

8. The DNA cassette for producing CD34 positive hematopoietic stem cells transfected with one or more copies of globin gene as claimed in claim 1 wherein, the therapeutic assembly can be contained within DNA minicircles without a transposon so that the patient can be treated with a simple injection of purified DNA minicircle provided to the patient in one or multiple settings (single injection or repeated injections) till complete symptomatic relief is obtained from the disease.

9. The DNA cassette for producing CD34 positive hematopoietic stem cells transfected with one or more copies of globin gene as claimed in claim 1 wherein the cassette can be used for the treatment of diseases related to red blood cells and other gene deficiencies where the hemoglobin gene can be replaced by the therapeutic gene for the respective disease. , Description:Before the present invention is described, it is to be understood that this invention is not limited to particular methodologies described, as these may vary as per the person skilled in the art. It is also to be understood that the terminology used in the description is for the purpose of describing the particular embodiments only, and is not intended to limit the scope of the present invention. Throughout this specification, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. The use of the expression “at least” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the invention to achieve one or more of the desired objects or results. The present invention describes a DNA cassette for the constitutive or inducible expression of the therapeutic genes within the selected carrier cells. The said DNA cassette comprises of therapeutic hemoglobin gene sequence with or without herpes simplex viral thymidine kinase (HSV-tk) and cytosine deaminase (CD) as suicidal switch genes along with Guassia luciferase or any other kind of luciferase gene. The said DNA cassette consisting of therapeutic genes either alone or in combination is incorporated into the carrier cells of choice by use of an established plasmid. The DNA cassette can be inserted into a bacterial plasmid or viral particles during the process of propagation and transduction. Bacterial culture can be used for multiplication of Plasmid number. Once expressed, the therapeutic genes will be available for an extended but limited period of time at the site of injury providing optimal clinical benefit. Further, as the carrier cells are appropriately transfected with the said DNA cassette, the cells will be intravenously administered to patient. Two populations of said carrier cells expressing, individual genes like luciferase and suicidal genes can also be infused to the patient. In another embodiment of the present invention, the said DNA cassette can be tagged with the expression of Bleomycin/ Zeocin, Green fluorescent protein or small engineered DNA tag namely His/FLAG tag can be added to therapeutic genes to confirm the functional expression of the said DNA cassette visually by Green fluorescent protein or Mass Spectrometry or ELISA or Western Blot. The novelty of the present invention lies in the therapeutic mechanism offered by ingenuously allowing the CD34+ cells expressing luciferase gene, suicidal genes along with the expression of beta globin gene expressed under an erythroid specific promoter. The DNA cassette can be used for gene therapy involving direct delivery of genes by use of lentiviral vector or it can be transfected to the CD34+ cells by use of other methods like lipofectamine and/or electroporation. Similarly, CD34+ promoter can be used to express the globin gene using minicircle technology. Non-baceterial supercoiled DNA minicircles can also be used to generate effective and extremely safe therapy for a period of 6 months to 1 year because this kind of treatment should not require any conditioning regimen or immunosuppressant therapy. The expression level can be monitored by taking blood and urine samples of patients for measuring the luciferase activity and the cell growth and death can be controlled by virtue of a suicidal switch. In one embodiment of this invention, multiple copies of beta globin gene are introduced so as to increase levels of expression driven under the erythroid specific promoter KLF-1. In another embodiment of this invention, the DNA construct includes 2 copies of beta globin and two suicide genes; cytosine deaminase and 5-fluorouracil. In yet another embodiment of this invention, the normal betaglobin gene copy and reporter gene with or without the suicide gene are bundled in a DNA minicircle to express the gene copies under an erythroid specific promoter.

Claims:1. A DNA cassette for producing CD34 positive hematopoietic stem cells transfected with one or more copies of globin gene, one or more suicide genes including HSV-tk and Cytosine deaminase (but not exclusively these 2 suicide genes) expressed either individually or in combination such that both sequences are flanking the two sides of viral 2A peptide or internal ribosomal entry (IRES) sequence.
2. The DNA cassette for producing CD34 positive hematopoietic stem cells transfected with one or more copies of globin gene, as claimed in claim 1 wherein, the said therapeutic genes are expressed under any known conventional promoter, including but not exclusively an erythroid specific promoter like KLF-1 promoter.
3. The DNA cassette for producing CD34 positive hematopoietic stem cells transfected with one or more copies of globin gene as claimed in claim 1 wherein, the said therapeutic genes are expressed along with luciferase gene for easy monitoring of the transgenic cells and or along with erythropoietin gene to increase the number of red blood cells.
4. The DNA cassette for producing CD34 positive hematopoietic stem cells transfected with one or more copies of globin gene as claimed in claim 1 wherein, the said carrier cells utilized for expressing the said DNA cassette to form the normal red blood cells (RBCs), and thus treat and cure the hemoglobin related diseases in patients of thalassemia, sickle cell disease and other genetic diseases.
5. The DNA cassette for producing CD34 positive hematopoietic stem cells transfected with one or more copies of globin gene as claimed in claim 1, wherein the said DNA cassette allows constitutive or inducible expression of the said therapeutic genes within the selected carrier cells for producing normal red blood cells.
6. The DNA cassette for producing CD34 positive hematopoietic stem cells transfected with one or more copies of globin gene as claimed in claim 1 wherein, the said the DNA cassette is tagged with the appropriate selection marker like Bleomycin/ Zeocin, Green fluorescent protein or any small DNA tag (His/FLAG tag etc. to confirm the functional expression of the said DNA cassette visually.
7. The DNA cassette for producing CD34 positive hematopoietic stem cells transfected with one or more copies of globin gene as claimed in claim 1 wherein, the therapeutic assembly can be contained within the inverted terminal repeats of transposons so as to provide permanent expression in cells without use of viral mode of transfection and use of DNA minicircles so that the patients can be treated with a simple intravenous injection or infusion provided to the patient in one or multiple settings (single injection or repeated injections) till complete symptomatic relief is obtained from the disease.
8. The DNA cassette for producing CD34 positive hematopoietic stem cells transfected with one or more copies of globin gene as claimed in claim 1 wherein, the therapeutic assembly can be contained within DNA minicircles without a transposon so that the patient can be treated with a simple injection of purified DNA minicircle provided to the patient in one or multiple settings (single injection or repeated injections) till complete symptomatic relief is obtained from the disease.
9. The DNA cassette for producing CD34 positive hematopoietic stem cells transfected with one or more copies of globin gene as claimed in claim 1 wherein the cassette can be used for the treatment of diseases related to red blood cells and other gene deficiencies where the hemoglobin gene can be replaced by the therapeutic gene for the respective disease.
, Description:Before the present invention is described, it is to be understood that this invention is not limited to particular methodologies described, as these may vary as per the person skilled in the art. It is also to be understood that the terminology used in the description is for the purpose of describing the particular embodiments only, and is not intended to limit the scope of the present invention. Throughout this specification, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. The use of the expression “at least” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the invention to achieve one or more of the desired objects or results.
The present invention describes a DNA cassette for the constitutive or inducible expression of the therapeutic genes within the selected carrier cells. The said DNA cassette comprises of therapeutic hemoglobin gene sequence with or without herpes simplex viral thymidine kinase (HSV-tk) and cytosine deaminase (CD) as suicidal switch genes along with Guassia luciferase or any other kind of luciferase gene.
The said DNA cassette consisting of therapeutic genes either alone or in combination is incorporated into the carrier cells of choice by use of an established plasmid. The DNA cassette can be inserted into a bacterial plasmid or viral particles during the process of propagation and transduction. Bacterial culture can be used for multiplication of Plasmid number.
Once expressed, the therapeutic genes will be available for an extended but limited period of time at the site of injury providing optimal clinical benefit. Further, as the carrier cells are appropriately transfected with the said DNA cassette, the cells will be intravenously administered to patient. Two populations of said carrier cells expressing, individual genes like luciferase and suicidal genes can also be infused to the patient.
In another embodiment of the present invention, the said DNA cassette can be tagged with the expression of Bleomycin/ Zeocin, Green fluorescent protein or small engineered DNA tag namely His/FLAG tag can be added to therapeutic genes to confirm the functional expression of the said DNA cassette visually by Green fluorescent protein or Mass Spectrometry or ELISA or Western Blot.
The novelty of the present invention lies in the therapeutic mechanism offered by ingenuously allowing the CD34+ cells expressing luciferase gene, suicidal genes along with the expression of beta globin gene expressed under an erythroid specific promoter.
The DNA cassette can be used for gene therapy involving direct delivery of genes by use of lentiviral vector or it can be transfected to the CD34+ cells by use of other methods like lipofectamine and/or electroporation. Similarly, CD34+ promoter can be used to express the globin gene using minicircle technology. Non-baceterial supercoiled DNA minicircles can also be used to generate effective and extremely safe therapy for a period of 6 months to 1 year because this kind of treatment should not require any conditioning regimen or immunosuppressant therapy.
The expression level can be monitored by taking blood and urine samples of patients for measuring the luciferase activity and the cell growth and death can be controlled by virtue of a suicidal switch.
In one embodiment of this invention, multiple copies of beta globin gene are introduced so as to increase levels of expression driven under the erythroid specific promoter KLF-1.
In another embodiment of this invention, the DNA construct includes 2 copies of beta globin and two suicide genes; cytosine deaminase and 5-fluorouracil.
In yet another embodiment of this invention, the normal betaglobin gene copy and reporter gene with or without the suicide gene are bundled in a DNA minicircle to express the gene copies under an erythroid specific promoter.