Optimization of determinants for successful genetic correction of diseases, mediated by hematopoietic stem cells

Abstract

Methods and compositions disclosed herein generally relates to methods of determining minimum hematopoietic stem cell (HSC) chimerism and gene dosage for correction of a hematopoietic disease; in particular, in in vivo models. The invention also relates to modified lentiviral expression vectors for increase a viral titer and various methods for increasing such titers as well as expression vectors capable of enhancing such titers. The invention also relates to CHS4 chromatin insulator-derived functional insulator sequences. The invention further relates to methods for genetic correction of diseases or reducing symptoms thereof, such as sickle cell anemia, a lysosomal storage disease. The invention further relates to a method of improving and/or correcting one or more central nervous system (CNS) abnormalities caused by one or more lysosomal storage disease. The invention further relates to methods of improving titer in transfection-based bioreactor culture production or transfection-based production systems using eukaryotic cells.

1 .- 26 . (canceled) 
     
     
         27 . A method of genetically modifying an HSC offspring cell to produce a protein, comprising:
 transducing an HSC with a vector, the vector comprising:
 an HSC offspring specific promoter; and 
 a gene of interest (GOI) operably linked to the promoter, the GOI encoding the protein, 
   wherein activation of the HSC offspring specific promoter leads to expression by the HSC offspring cell of the protein.   
     
     
         28 . The method of  claim 27 , wherein the HSC offspring cell forms an anuclear cell. 
     
     
         29 . The method of  claim 27 , wherein the HSC offspring cell is a maturing erythroid cell. 
     
     
         30 . The method of  claim 27 , wherein the protein is alpha-L-iduronidase (IDUA). 
     
     
         31 . The method of  claim 27 , wherein the protein is non-native to blood, is not conventionally secreted into blood circulation, or both. 
     
     
         32 . The method of  claim 27 , wherein activation of the HSC offspring specific promoter leads to expulsion by the HSC offspring cell of the protein. 
     
     
         33 . A method of treating an individual for Mucopolysaccharidosis type I (MPS I), reducing symptoms thereof, or both, comprising:
 introducing an HSC into an individual with MPS I, wherein the expression of IDUA from an HSC offspring cell leads to increased IDUA levels in the individual's blood, and results in treatment of MPS I, a reduction of symptoms thereof, or both,   wherein the HSC is transduced with a vector, the vector comprising (a) an HSC offspring specific promoter and (b) a gene encoding IDUA,   wherein activation of the HSC offspring specific promoter leads to the expression of IDUA by the HSC offspring cell.   
     
     
         34 . The method of  claim 33 , wherein the HSC offspring cell forms an anuclear cell. 
     
     
         35 . The method of  claim 33 , wherein the HSC offspring cell is a maturing erythroid cell. 
     
     
         36 . The method of  claim 33 , wherein (a) the lysosomal storage pathology in the individual is corrected in the liver, spleen, or both, (b) the individual has neurological improvement, or (c) both. 
     
     
         37 . The method of  claim 33 , wherein activation of the HSC offspring specific promoter leads to the expulsion of IDUA by the HSC offspring cell. 
     
     
         38 . An expression vector capable of treating MPS type I, reducing symptoms thereof, or both, comprising:
 an HSC offspring specific promoter; and   a gene encoding IDUA, wherein activation of the HSC offspring specific promoter leads to expression of IDUA by an HSC offspring cell.   
     
     
         39 . The expression vector of  claim 38 , wherein the HSC offspring cell forms an anuclear cell. 
     
     
         40 . The expression vector of  claim 38 , wherein the HSC offspring cell is a maturing erythroid cell. 
     
     
         41 . The expression vector of  claim 38 , wherein activation of the HSC offspring specific promoter leads to expulsion of IDUA by an HSC offspring cell. 
     
     
         42 . An expression vector capable of treating or reducing symptoms of a disease characterized by insufficient expression of a least one functional protein, comprising:
 an HSC offspring specific promoter; and   a GOI encoding a protein,   
       wherein activation of the HSC offspring specific promoter leads to the expression by an HSC offspring cell of the protein. 
     
     
         43 . The expression vector of  claim 42 , wherein the HSC offspring cell forms an anuclear cell. 
     
     
         44 . The expression vector of  claim 42 , wherein the HSC offspring cell is a maturing erythroid cell. 
     
     
         45 . The expression vector of  claim 42 , wherein the protein is non-native to blood, is not conventionally secreted into blood circulation, or both. 
     
     
         46 . The expression vector of  claim 42 , wherein the protein is IDUA. 
     
     
         47 . The expression vector of  claim 42 , wherein activation of the HSC offspring specific promoter leads to the expulsion by an HSC offspring cell of at least one protein. 
     
     
         48 . A method of treating an individual with a lysosomal storage disease characterized by deficiency of one or more lysosomal enzymes, comprising:
 introducing an HSC into the individual with the lysosomal storage disease, wherein the expression of one or more lysosomal enzymes from an HSC offspring of a HSC provides increased lysosomal enzyme level(s) in the individual's blood, resulting in correction of the lysosomal storage disease, a reduction of symptoms thereof, or both,   wherein the HSC is transduced with a vector comprising (a) an HSC offspring specific promoter; and (b) a gene encoding one or more lysosomal enzymes,   wherein activation of the HSC offspring specific promoter leads to the expression of a lysosomal enzyme by an HSC offspring cell.   
     
     
         49 . The method of  claim 48 , wherein one or more of the sustained lysosomal enzyme level(s) in the individual's blood is normal or high compared to a person without the lysosomal storage disease. 
     
     
         50 . The method of  claim 48 , wherein one or more lysosomal enzymes are endocytosed into cells through mannose 6-phosphate mediated endocytosis. 
     
     
         51 . The method of  claim 48 , wherein the HSC offspring cell forms an anuclear cell. 
     
     
         52 . The method of  claim 48 , wherein the HSC offspring cell is a maturing erythroid cell. 
     
     
         53 . The method of  claim 48 , wherein the at least one lysosomal enzyme is IDUA. 
     
     
         54 . The method of  claim 48 , wherein (a) the lysosomal storage pathology in the individual is corrected in the liver, spleen, or both, (b) the individual has neurological improvement, or (c) both. 
     
     
         55 . The method of  claim 48 , wherein activation of the HSC offspring specific promoter leads to the expulsion of a lysosomal enzyme by an HSC offspring cell. 
     
     
         56 . A method of treating one or more central nervous system (CNS) abnormalities caused by one or more lysosomal storage diseases, comprising:
 introducing an HSC into an individual with a lysosomal storage disease, wherein the expression of one or more lysosomal enzyme(s) from an HSC offspring of the HSC provides increased lysosomal enzyme level(s) in the individual's blood stream, and correction or improvement of one or more CNS abnormalities caused by one or more lysosomal storage diseases,   wherein the HSC is transduced with a vector, the vector comprising (a) an HSC offspring specific promoter and (b) a gene encoding one or more lysosomal enzymes,   wherein activation of the HSC offspring specific promoter leads to the expression of one or more lysosomal enzymes by an HSC offspring cell.   
     
     
         57 . The method of  claim 56 , wherein one or more of the sustained lysosomal enzyme level(s) in the individual's blood is normal or higher compared to an individual without the lysosomal storage disease. 
     
     
         58 . The method of  claim 56 , wherein the HSC offspring cell forms an anuclear cell. 
     
     
         59 . The method of  claim 56 , wherein the HSC offspring cell is a maturing erythroid cell. 
     
     
         60 . The method of  claim 56 , wherein the at least one lysosomal enzyme is IDUA. 
     
     
         61 . The method of  claim 56 , wherein (a) the lysosomal storage pathology in the individual is corrected in the liver, spleen, or both, (b) the individual has neurological improvement, or (c) both. 
     
     
         62 . The method of  claim 56 , wherein activation of the HSC offspring specific promoter leads to the expulsion of one or more lysosomal enzymes by an HSC offspring cell.