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

a technology of hematopoietic stem cells and determinants, which is applied in the field of optimizing determinants for successful genetic correction of diseases, can solve the problems of reducing titers, limited treatment options for lsds, and little to address cns pathologies for this group of disorders, so as to enhance the stability and safety of gene expression and enhance the stability and safety of transgene expression

Inactive Publication Date: 2015-11-05
CHILDRENS HOSPITAL MEDICAL CENT CINCINNATI
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AI Technical Summary

Benefits of technology

The patent text describes a modified lentiviral vector that has improved viral titer and stability compared to a standard vector. The vector includes a gutted / minimal backbone devoid of certain elements and a therapeutic transgene of interest. The vector also contains a heterologous polyA signal sequence or a USE sequence derived from an SV40 late polyA signal in the vector backbone. The vector may also include flanking cHS4-derived reduced-length functional insulator sequences, a lineage-specific promoter and / or enhancer, and one or more lentivirus non-coding cis sequences. The technical effects of the modified vector include improved viral titer, reduced transcriptional read-through of vector transcripts, improved packaging efficiency, and increased stability and safety of gene expression.

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.

Method used

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

Examples

Experimental program
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Effect test

example 1

Lentivirus Cis Elements Required for Efficient Packaging of Large Transgenes Cassettes Like β-Globin

[0117]This study investigated whether lentivirus non-coding cis-sequences played a specific role in the RNA export, packaging or expression of β-globin. The vector life-cycle was studied in self-inactivating (SIN)-lentiviruses, carrying the β-globin gene and locus control region (BG), or GFP cDNA. Systematic analysis started with a completely ‘gutted’ minimal SIN-lentivirus carrying only the packaging region; and SIN-lentiviruses containing increasing HIV cis-elements, along with a SIN-gamma-retrovirus. It was discovered that (i) SIN-gamma-retrovirus or a gutted / minimal SIN-lentivirus encoding GFP generated high titers and mediated high GFP expression. (ii) However, SIN-gamma-retrovirus or the gutted SIN-lentivirus encoding either BG or a similar sized large transgene had barely detectable titers compared to the SIN-lentivirus carrying cis-elements. (iii) Systematic addition of cis-el...

example 2

BG Expression from Gutted SIN-γRV

[0118]It has been postulated that γRV are unable to successfully express hβ-globin due to transcriptional interference between the strong γRV LTR promoter / enhancer elements and the internal LCR enhancer. SRS11.SF is a SIN-γRV that encodes the GFP cDNA under control of an internal Spleen Focus-Forming Virus (SFFV) promoter / enhancer. The SFFV-GFP in SRS11.SF was replaced with BG, an expression cassette that was successfully utilized in a standard SIN-LV to achieve therapeutic hβ-globin expression in thalassemia, to generate SRS11.BG. The rationale for using SRS.11, despite the notoriety of β-globin γRV was: (i) it contains the minimal packaging region (ψ), lacks gag sequences and can carry a larger vector payload, yet retains extremely high titers; (ii) it carries a large 400 bp U3 deletion of the 3′LTR, comparable to the deletion in SIN-LV. (iii) Large LCR elements have never been tested in γRV due to restrictions on vector payload.

[0119]Infectious ti...

example 3

Expression of Large / Small Transgenes from Standard or Gutted / Minimal LV

[0120]In contrast to the SIN-γRV used herein, the “standard” SIN-LV commonly used retains relatively large portions of viral sequences amounting to about 20-25% of the HIV genome. These cis elements are: the LTR (634 bp for wt HIV LTR or 235 bp for SIN-LV LTR), the packaging signal ψ(150 bp), 5′ portion of the gag gene (300 or 600 bp), env sequences including the rev response element (RRE, 840 bp) and the central flap / polypurine tract (cPPT) from the pol gene (120 bp).

[0121]To examine the requirement of cis-sequences for GFP versus BG, the CMV-GFP cassette was cloned in a) the “standard” SIN-LV containing cis sequences listed above (sSIN-GFP), and b) a ‘gutted’ minimal SIN-LV where the gag, RRE and the rest of the env sequences were deleted and only the ψ region was retained (dsSIN-GFP; FIG. 1A). The titers of the minimal dsSIN-GFP LV were only 2-times lower than the titers of the “standard” LV sSIN-GFP FIG. 1B; ...

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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.

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...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/86
CPCC12N15/86C12N2830/48C12N2740/15043C12N15/63C12N15/85
Inventor VAN DER LOO, JOHANNES CHRISTIAAN MARIAPAN, DAOMALIK, PUNAM
Owner CHILDRENS HOSPITAL MEDICAL CENT CINCINNATI
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