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 . A method of determining minimum hematopoietic stem cell (HSC) chimerism and gene dosage for correction of a hematopoietic disease in an in vivo model, comprising:
 inducing various levels of chimerism and gene dosage post-transplantation, wherein various levels of chimerism and gene dosage are induced by applying reduced intensity conditioning prior to transplantation;   transducing cells at a range of multiplicity of infection (MOI), wherein MOI is 30-100; and   inducing varying levels of chimerism and gene dosage results in determining minimum HSC chimerism and gene dosage for correction of a hematopoietic disease in an in vivo model.   
     
     
         2 . A modified SIN lentiviral expression vector capable of enhanced viral titer in comparison with a standard SIN lentiviral expression vector, comprising:
 a gutted/minimal Cis lentiviral vector backbone devoid of Cis elements, except the packaging signal (ψ); and   a small therapeutic transgene of interest (GOI), wherein a modified SIN lentiviral expression vector containing devoid of Cis elements, except a ψ Cis element, enhances viral titer in comparison with a standard SIN lentiviral expression vector.   
     
     
         3 . A CHS4 chromatin insulator-derived functional insulator sequence, comprising:
 a proximal core region of the CHS4 insulator sequence comprising 250 base pairs (bp) or less; and   a distal element of the CHS4 insulator sequence, comprising 400 bp or less, wherein the functional insulator sequence is at least 80% as effective as an unmodified CHS4 chromatin insulator, and wherein the functional insulator sequence permits a higher titer of expression of a vector carrying such a sequence, in comparison with a vector carrying the unmodified CHS4 chromatin insulator.   
     
     
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