Stable Gene Transfer to Proliferating Cells

a technology of proliferating cells and gene transfer, applied in the direction of transferases, ligases, genetic material ingredients, etc., can solve the problems of hammering the further development of aav-based gene therapy approaches, and achieve the effect of stable integration and expression of transgenes

Inactive Publication Date: 2017-08-03
MOUNT SINAI HOSPITAL +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0013]A third aspect of the invention provides a method for treating or preventing a disease of, affecting, or associated with, a proliferating cell, comprising administering to a subject in need thereof (i) a recombinant AAV (rAAV) vector comprising a transgene flanked by transposon-derived inverted terminal repeat sequences, which sequences are in turn flanked by AAV

Problems solved by technology

Notwithstanding the attractive aspects of AAV-based vectors, a significant challenge, as yet not overcome, to their widespread use is maintaining stable levels of therapeutically effective transgene expression in proliferating cells suc

Method used

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  • Stable Gene Transfer to Proliferating Cells
  • Stable Gene Transfer to Proliferating Cells
  • Stable Gene Transfer to Proliferating Cells

Examples

Experimental program
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Effect test

example 1

posase Vector Constructs

[0070]Transposon-donor vectors and a piggyBac Transposase vector were constructed using the recombinant adeno-associated viral vector (rAAV) system. The hybrid AAV / transposase system was subsequently used (see Examples 2 to 4) to demonstrate phenotype correction in animal models with genetic metabolic disease phenotypes. These included the spfash mouse model of ornithine transcarbamylase (OTC) deficiency and the citrullinaemic mouse model of argininosuccinate synthetase (ASS) deficiency (both urea cycle disorders), and the PFIC3 mouse model (ABCB4 deficiency) of progressive familial intrahepatic cholestasis. Each of these disease phenotypes presents early in life, in neonates or juveniles.

[0071]The coding sequence of piggyBac transposase was amplified by PCR from pCAG-PBase. The piggyBac transposase vector was constructed by inserting the coding region of the piggyBac transposase into a rAAV2 genome under the transcriptional control of a liver-specific promot...

example 2

pression of Hybrid AAV / Transposase Constructs in Mice

[0131]The ability of the AAV / transposase vector systems described in Example 1 to stably integrate and express a transgene in a host genome was determined using transposon-donor vectors encoding enhanced green fluorescent protein (EGFP) administered to C3H and FVB.129P2-Abcb4tm1Bor mice. Animals were housed in a temperature-controlled environment with 12-hour light / dark cycles with water and standard rodent chow (18.9% (wt / wt) protein; Specialty Feeds, Glen Forrest, Australia) supplied ad libitum. All experimental procedures were evaluated and approved by the institutional Animal Care and Ethics Committee. The experimental design is outlined in FIG. 2. Four mice were used for each group. Constructs were administered by injection via the intraperitoneal route in 20 μL volumes (diluted in PBS with calcium and magnesium) in newborn mice, at vector doses of 5×1010 vg / mouse for the transposase vector, and 1×1011-5×1011 vg / mouse for the...

example 3

apy in a Mouse Model of OTC Deficiency

[0138]The hybrid AAV / transposase constructs described in Example 1 were used to demonstrate phenotype correction in a mouse model of OTC deficiency (the spfash mouse model). Mice used were strain B6EiC3Sn a / A-Otcspf-ash / J (provided by The Jackson Laboratory). The disease phenotype presents early in life in neonates or juveniles. As such, vector treatment was delivered to mice during the neonatal period. Constructs were administered by injection via the intraperitoneal route in 20 μL volumes (diluted in PBS with calcium and magnesium) in newborn mice (1-2 days), at vector doses of 5×1010 vg / mouse for the transposase vector, and 1×1011 vg / mouse for the transposon-transgene donor vector. The experimental design is outlined in FIG. 4 with 12 mice receiving OTC-transposon-encoding AAV2 / 8 vector alone, and 12 mice receiving OTC transposon-encoding vector in combination with the piggyBac transposase-encoding AAV2 / 8 vector.

[0139]Liver sections from mice...

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Abstract

Provided herein are methods for facilitating or inducing stable transgene integration and expression in a proliferating cell, comprising administering to the cell (i) a recombinant AAV (rAAV) vector comprising the transgene flanked by transposon-derived inverted terminal repeat sequences, which sequences are in turn flanked by AAV-derived inverted terminal repeat regions, and (ii) a source of a transposase that recognises said transposon-derived inverted terminal repeat sequences and directs the genomic integration of the transgene into the genome of the proliferating cell. Also provided are methods and transgene delivery systems for the treatment or prevention of diseases affecting, associated with or characterised by proliferating cells, including paediatric liver diseases, bone marrow diseases and cancer.

Description

TECHNICAL FIELD[0001]The present invention relates generally to methods for stably integrating and expressing transgenes in proliferating cells. The invention also relates to methods and vector systems for the treatment of genetic diseases associated with, or affecting, proliferating cells, organs or tissues.BACKGROUND ART[0002]Adeno-associated virus (AAV) is a parvovirus having a single-stranded DNA genome. The AAV genome is relatively simple, containing two open reading frames (ORFs) flanked by short inverted terminal repeats (ITRs). The ITRs contain, inter alia, cis-acting sequences required for virus replication, rescue, packaging and integration. The integration function of the ITR permits the AAV genome to integrate into a cellular chromosome after infection.[0003]Recombinant AAV vectors have been shown to be able to transduce a wide range of different cell types, such as hematopoietic cells, epithelial cells and neurons. Interest in AAVs as vectors for gene therapy results fr...

Claims

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Application Information

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IPC IPC(8): A61K48/00C12N9/12C12N9/00C12N9/14C12N15/86C12N9/10
CPCA61K48/0066A61K48/0041C12N15/86C12Y201/03003C12Y603/04005C12Y306/03044C12N2840/007C12N9/93C12N9/14C12Y207/07C12N9/1241C12N2750/14143C12N2800/90C12N9/1018A61K48/005
Inventor ALEXANDER, IANCUNNINGHAM, SHARONNAGY, ANDRAS
Owner MOUNT SINAI HOSPITAL
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