Optimization of determinants for successful genetic correction of diseases, mediated by hematopoietic stem cells
First Claim
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.
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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.
82 Citations
4 Claims
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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:
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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.
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2. A modified SIN lentiviral expression vector capable of enhanced viral titer in comparison with a standard SIN lentiviral expression vector, comprising:
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a gutted/minimal Cis lentiviral vector backbone devoid of Cis elements, except the packaging signal (ψ
); anda 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.
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3. A CHS4 chromatin insulator-derived functional insulator sequence, comprising:
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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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4-26. -26. (canceled)
Specification