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 futher 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 futher 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 modified self-inactivating (SIN) lentiviral vector, comprising:
 (a) a lentiviral vector backbone comprising:
 (1) lentiviral cis elements, which consist essentially of (i) a packaging signal (ψ), 
   (ii) a gag fragment, (iii) an env fragment containing a rev response element (RRE) and a splice acceptor sequence (SA), and (iv) a central polypurine tract (cPPT), wherein the gag fragment comprises a 5′ fragment of gag that is about 300 bp;   and   (b) a transgene of interest.   
     
     
         28 . The modified SIN lentiviral vector of claim  1 , wherein the gag fragment consists of the 5′ fragment of gag that is about 630 bp in length. 
     
     
         29 . The modified SIN lentiviral vector of claim  1 , wherein the gag fragment consists of the 5′ fragment of gag that is about 360 bp in length. 
     
     
         30 . The modified SIN lentiviral vector of claim  1 , wherein the lentiviral vector backbone of (a) further comprises:
 (2) a 3′-long terminal repeat (LTR), which comprises an upstream polyadenylation (polyA) enhancer signal sequence; and   (3) one or more copies of a heterologous polyA signal sequence downstream from the 3′-LTR;   
     
     
         31 . The modified SIN lentiviral vector of  claim 30 , wherein the heterologous polyA signal sequence is a13-growth hormone polyA signal sequence. 
     
     
         32 . The modified SIN lentiviral vector of  claim 30 , wherein the U3 region of the 3′-LTR is deleted. 
     
     
         33 . The modified SIN lentiviral vector of  claim 32 , wherein the U3 region is replaced with the upstream polyadenylation (polyA) enhancer signal sequence, which is an upstream sequence element (USE) derived from an SV40 late polyA signal sequence. 
     
     
         34 . The modified SIN lentiviral vector of  claim 27 , wherein the lentiviral vector backbone further comprises one or more chromatin insulator elements. 
     
     
         35 . The modified SIN lentiviral vector of  claim 34 , wherein the chromatin insulator elements include a chicken hypersensitive site-4 element (cHS4). 
     
     
         36 . The modified SIN lentiviral vector of  claim 35 , wherein the cHS4 element contains an about 250-base pair core sequence of a cHS4 insulator linked to a distal element of the cHS4, which is about 400 base pair or less. 
     
     
         37 . The modified SIN lentiviral vector of  claim 36 , wherein the cHS4 element is inside the 3′-LTR. 
     
     
         38 . The modified SIN lentiviral vector of  claim 27 , wherein the lentiviral cis elements consists essentially of the packaging signal (ψ), the env fragment, and the gag fragment, which consists of the 5′ fragment of gag that is about 360 bp, and wherein the lentiviral vector backbone further comprises an upstream sequence element (USE) derived from an SV40 late polyA signal sequence, a β-growth hormone polyA signal sequence, and one or more chicken hypersensitive site-4 elements (cHS4s). 
     
     
         39 . The modified SIN lentiviral vector of  claim 27 , wherein the transgene of interest is a gamma-globin gene. 
     
     
         40 . The modified SIN lentiviral vector of  claim 39 , wherein the gamma-globin gene is a human gamma-globin gene. 
     
     
         41 . The modified SIN lentiviral vector of  claim 39 , wherein the gamma-globin gene is in a reverse orientation to a viral transcription unit in the lentiviral vector backbone. 
     
     
         42 . The modified SIN lentiviral vector of claim  1 , wherein the transgene of interest is in operable linkage to a lineage-specific promoter. 
     
     
         43 . The modified SIN lentiviral vector of  claim 42 , wherein the transgene of interest is in further operable linkage to a lineage-specific enhancer. 
     
     
         44 . The modified SIN lentiviral vector of  claim 43 , wherein the enhancer is an erythroid lineage specific enhancer element and the transgene of interest is a gamma-globin gene. 
     
     
         45 . The modified SIN lentiviral vector of  claim 39 , wherein the gamma-globin gene is a hybrid gene comprising gamma-globin coding sequences and beta-globin non-coding and regulatory sequences. 
     
     
         46 . The modified SIN lentiviral vector of  claim 45 , wherein the beta-globin non-coding and regulatory sequences comprises one or more of HS2, HS3, and HS4 of the locus control region of beta-globin gene. 
     
     
         47 . The modified SIN lentiviral vector of  claim 46 , wherein the gamma-globin gene is under the control of the beta-globin regulatory sequences. 
     
     
         48 . A host cell comprising the modified SIN lentiviral vector of claim  1 . 
     
     
         49 . The host cell of  claim 48 , wherein the host cell is a hematopoietic stem cell. 
     
     
         50 . A method for preparing a transgenic host cell, comprising transfecting the modified SIN lentiviral vector of claim  1  into a host cell. 
     
     
         51 . A method for genetically correct sickle cell anemia or β-thalassemia or reduce symptoms thereof, the method comprising:
 providing the host cell of  claim 50 , wherein the host cell is a hematopoietic stem cell and wherein the transgene gene of interest in the modified SIN lentiviral vector is a gamma-globin gene, and 
 transplanting the host cell into a subject in need thereof.