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.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of U.S. application Ser. No. 12 / 928,302, filed on Dec. 6, 2010, and is also a continuation of U.S. application Ser. No. 13 / 946,746, filed on Jul. 19, 2013, which is a continuation of U.S. application Ser. No. 12 / 928,302, filed on Dec. 6, 2010, which claims priority from U.S. Provisional Application No. 61 / 267,008, filed on Dec. 4, 2009, the contents of each of which are herein incorporated by reference in their entirety.GOVERNMENT RIGHTS[0002]This invention was made with government support under HL073104, HL70135, HL070595, HL060008, HL079574, AI061703, and NS064330 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD OF THE INVENTION[0003]The invention 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 an in vivo model. T...

AI Technical Summary

Benefits of technology

[0014]In another aspect, a modified SIN lentiviral expression vector is provided, which is capable of enhanced viral titer in comparison with a standard SIN lentiviral expression vector. More specifically, the vector comprises: a gutted / minimal Cis lentiviral vector backbone devoid of Cis elements, except the packaging signal (ψ); and a small therapeutic transgene of interest (GOI), where the 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.

[0016]In another aspect, a method of increasing a titer of a modified SIN lentiviral vector, when compared to a standard SIN lentiviral vector, is provided. The method comprises: inserting one or more copies of a heterologous polyadenylation (polyA) signal sequence downstream from a viral 3′ long terminal repeat sequence in a standard SIN lentiviral vector backbone, resulting in a polyA-modified SIN lentiviral vector; and transfecting a eukaryotic cell with the polyA-modified vector, where insertion of the heterologous polyA signal sequence and subsequent transfection of the eukaryotic cell increases the viral titer of the modified vector, when compared to the standard SIN lentiviral vector. In some embodiments, the insertion of the heterologous polyA signal sequence and subsequent transfection of the eukaryotic cell reduces transcriptional read-through of vector transcripts, improves packaging efficiency and increases the viral titer of the modified vector, when compared to the standard SIN lentiviral vector.

[0017]In another aspect, a method of increasing titer of a modified SIN lentiviral vector, when compared to a standard SIN lentiviral vector, is provided. The method comprises: inserting one or more of an upstream polyA enhancer signal into a 3′ LTR of a standard SIN lentiviral vector backbone, resulting in a polyA enhancer-modified lentiviral vector; and transfecting a eukaryotic cell with the polyA enhancer-modified lentiviral vector, where transfection with the polyA enhancer-modified vector results in an increased titer as compared to titer of a standard SIN lentiviral vector. In some embodiments, the polyA enhancer is a USE sequence derived from an SV40 late polyA signal. In some embodiments, transfection with the USE sequence derived vector increases titer by reducing transcriptional read-through of vector transcripts and improving packaging efficiency. In some embodiments, three upstream polyA enhancer signal sequences are inserted into the 3′ LTR of a standard SIN lentiviral vector backbone.

[0020]In another aspect, an expression vector capable of enhancing stability and safety of gene expression while maintaining a clinically useful titer of a lentiviral vector is provided. The expression vector comprises: a heterologous polyA signal sequence downstream from a viral 3′ LTR sequence in a standard SIN lentiviral vector backbone; a USE sequence derived from an SV40 late polyA signal in a U3 deletion region of a standard SIN lentiviral vector backbone; one or more flanking cHS4-derived reduced-length functional insulator sequences; and a lineage-specific promoter and / or enhancer selected for restricted activation in cells in which expression is desired.

[0021]In another aspect, an expression vector capable of enhancing stability and safety of transgene expression while maintaining a clinically useful titer of a lentiviral vector is provided. The expression vector comprises: a transgene of interest; a heterologous polyA signal sequence downstream from a viral 3′ LTR sequence in a standard SIN lentiviral vector backbone; a USE sequence derived from an SV40 late polyA signal in a U3 deletion region of a standard SIN lentiviral vector backbone; one or more flanking cHS4-derived reduced-length functional insulator sequences; a lineage-specific promoter and / or enhancer selected for restricted activation in cells in which expression is desired; and one or more lentivirus non-coding cis sequences, selected from: R, U5, a packaging signal, rev response element, env splice acceptor site, and an extended gag sequence.

Problems solved by technology

Although significant advances in vector design have improved the efficacy of gene therapy, certain key obstacles have emerged as barriers to successful clinical application.

While these insulators can improve the safety and expression profiles of certain vectors, in some cases an undesirable side effect is decreased titers compared to non-inactivated versions.

Treatment modalities for LSDs are currently limited to bone marrow transplantation (BMT) and enzyme replacement therapy (ERT).

These approaches while providing significant promise for treatment of the visceral manifestations of LSDs, do little to address CNS pathologies for this group of disorders.

Moreover, BMT is limited by procedure-related mortality between 20 and 30%, late complications such as graft versus host disease, and by the need to find an HLA-matched donor.

However, it is limited by poor penetration of the CNS, the need for frequent intravenous infusion for a lifetime and by tremendous costs.

Expressing a tremendous amount of fetal / antisickling hemoglobin will undoubtedly correct disease, as has been demonstrated, but is not practically possible in a clinical setting.

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