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Vcn enhancer compositions and methods of using the same

A technology of promoters and elongation factors, applied in biochemical equipment and methods, drug combinations, and the use of vectors to introduce foreign genetic materials, etc., can solve the problem of difficult and effective transduction of hematopoietic stem cells and progenitor cells, cell toxicity, and increased inefficient transduction Gene therapy costs and other issues

Pending Publication Date: 2018-10-26
BLUEBIRD BIO INC
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of these strategies, such as polycations, cationic liposomes, polybrene, involve the use of adjuvant therapies that are cytotoxic, limiting them to sensitive target cells of primary origin, such as hematopoietic Stem and Progenitor Cells Used Together
[0013] Hematopoietic stem and progenitor cells are known to be difficult to efficiently transduce, thus inefficient transduction is one of the major limiting factors preventing HSC-based gene therapy from entering the clinic
Inefficient transduction also increases the cost of developing gene therapy, as large numbers of vectors are required to generate the necessary amount of transduced cells

Method used

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  • Vcn enhancer compositions and methods of using the same
  • Vcn enhancer compositions and methods of using the same
  • Vcn enhancer compositions and methods of using the same

Examples

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Effect test

example 1

[0761] Validation analysis

[0762] Vector copy number (VCN) and transduction efficiency

[0763] Washed cells were resuspended in 2 mL of Stem Cell Growth Medium (SCGM) + cytokines and transferred to standard 12-well non-adherent tissue culture plates. Cells were maintained in a standard humidified tissue culture incubator (5% CO2) for an additional 6 days prior to vector copy number (VCN) analysis or single cell nested PCR to determine transduction efficiency. Both VCN and single-cell nested PCR analysis were performed by qPCR using primers and probes specific for the vector and endogenous control genes. VCN was determined by dividing the amount of vector signal by the amount of endogenous control gene. For single-cell nested PCR analysis, count wells containing cells positive for the endogenous control gene and wells containing labeled cells positive for both the vector and the endogenous control gene, and calculate the proportion of labeled cells or transduction efficien...

example 2

[0771] After PGE 2 processed human CD34 + In vivo analysis of cells

[0772] Contains PGE from healthy donors 2 Meta-analysis of NSG grafts of treated and lentivirally transduced hCD34+ cells (n=3). Human CD34 + Cells were transplanted into submyeloablative female NSG mice and bone marrow (BM) analyzed 4 months after transplantation. BM was harvested from both femurs of NSG mice and half of the samples were stained with anti-hCD45 for approximately 30 min at 4 °C. Following incubation, samples were washed and analyzed for human CD45 by flow analysis using an anti-hCD45 antibody (BD#561864) + cell. In control-treated samples versus PGE-treated 2 hCD45 in mouse BM was not detected between treated samples + The percentages of cells are statistically significantly different. These results indicated that hCD34 + Cellular PGE 2 Treatment did not significantly increase HSC engraftment. figure 1 a. At 4 months after transplantation, the transplanted 2 processed hCD34 +...

example 3

[0774] hCD34+ cells transduced in the presence of increasing concentrations of F108 showed dose-dependent increases in viral entry and VCN

[0775] lentiviral transduction of hCD34 in the presence of protamine sulfate or increasing concentrations of F108 + Cells for 2 hours or 24 hours. Analysis of F108 for lentiviral entry into human CD34 using the BlaM assay + Effects on cells. Cells were transduced with a lentiviral vector encoding a β-lactamase-Vpr fusion protein. After 2 hours of transduction, cells were washed and incubated with a fluorogenic substrate for β-lactamase. Cells were then analyzed by flow cytometry for β-lactamase substrate cleavage, which is associated with lentiviral entry into CD34 + cell related. Flow cytometry analysis revealed that increasing concentrations of F108 during transduction increased LVV entry into hCD34 + cells, an increased percentage of cells with cleaved BlaM substrate was observed. The effect of increased lentiviral entry appeare...

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Abstract

The invention provides improved gene therapy methods and compositions.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of the following applications under 35 U.S.C. §119(e): U.S. Provisional Application Nos. 62 / 429,514 filed December 2, 2016, 62 / 417,085 filed November 3, 2016, March 2016 US Provisional Application No. 62 / 313,571, filed on February 25, and US Provisional Application No. 62 / 294,615, filed February 12, 2016, each of which is incorporated herein by reference in its entirety. technical field [0003] The present invention relates in major part to improved gene therapy compositions and methods for their manufacture. Background technique [0004] Gene therapy offers great potential for a new era of human medicine. Gene therapy approaches can treat conditions that cannot be addressed by standard medical practice. However, the Food and Drug Administration (FDA) still has not approved any human gene therapy products for sale. Current gene therapy is experimental and has had mixed results i...

Claims

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Application Information

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IPC IPC(8): C12N5/078C12N5/074
CPCA61K35/28C12N5/0647C12N2510/00A61P25/00A61P25/02A61P25/08A61P25/28A61P37/02A61P5/38A61P7/00A61P7/06C07K14/7155C12N15/86C12N2740/15041C12Y301/06013C12Y302/01076C12Y304/14009C12Y305/04004C12N5/10
Inventor 梅丽莎·邦纳奥利维尔·尼格拉
Owner BLUEBIRD BIO INC
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