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Regulated expression of transgenes in the central nervous system of mammals

A technology of transgene and transcription factor, applied in the field of gene regulation

Inactive Publication Date: 2008-07-02
GENZYME CORP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although regulation of gene expression may not be necessary in the context of AADC therapy, since dopamine production is constitutively controlled by exogenous delivery of its precursor levodopa, regulation may be beneficial to precisely control the delivery of a dose of the enzyme or cause treatment discontinuation, if required

Method used

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  • Regulated expression of transgenes in the central nervous system of mammals
  • Regulated expression of transgenes in the central nervous system of mammals
  • Regulated expression of transgenes in the central nervous system of mammals

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0131] Regulatable expression of AADC in vitro

[0132] In vitro regulation of the AADC transgene was obtained as follows:

[0133] AAV vector

[0134] To obtain dimer-dependent expression of hAADC: AAV-CMV-TF and AAV-Z12-hAADC, two vectors were constructed. Figure 1A is a map of AAV-CMV-TF in which a cytomegalovirus (CMV) enhancer / promoter drives expression of a bicistronic message encoding an activation domain and DNA binding domain fusion protein. The activation domain fusion protein contains the human FRAP rapamycin binding domain (FRB ＊ ), which is fused to the (p65) transcriptional activation domain derived from NFκB. The particular FRB domain used in this embodiment (FRB T2098L ) and the specific FRB domain (FRB ＊ ) carries a T to L mutation at position 2098. Pollock et al. (2000) Proc. Natl. Acad. Sci USA 97:13221-26. FRB T2098L AP21967 or rapamycin can be used to dimerize to DNA-binding domain fusions.

[0135] The internal ribosome entry sequence (IRES) is d...

Embodiment 2

[0147] Tunable expression of AADC in vivo

[0148] In vivo assays were also used in the 6-hydroxydopamine (6-OHDA) rat model of Parkinson's disease to demonstrate AAV-mediated transduction of dimer-regulated hAADCs in vitro (Example 1, Figures 1A and 1B) The same vector, as follows. In Example 2, rapamycin was used instead of AP21667 as the dimer because of its higher potency and known pharmacokinetics. In the body, rapamycin has a half-life of approximately 10 hours and is rapidly cleared. Gallant-Haidner et al. (2000) Ther. Drug Monit. 22:31-5.

[0149] All rats used in this example had unilateral 6-OHDA lesions on the left side. There were three test groups: vehicle-infused control group (vehicle-infused (+) rapamycin-treated), vehicle-infused (-) rapamycin-treated control group and vehicle-infused (+) rapamycin-treated group . The vector infused (-) rapamycin group serves to control hAADC gene expression in the absence of rapamycin, ie, to determine if the system is l...

Embodiment 3

[0187] In vitro regulated expression of GDNF

[0188] Mammalian cells were transduced with dimer-regulatable GDNF transgene constructs, as follows.

[0189] AAV-GDNF vector

[0190] Figures 8A and 8B are diagrams of recombinant AAV vector plasmid constructs for delivery of the human GDNF (hGDNF) transgene. A control vector for delivery of constitutively expressed hGDNF (pAAV-CMV-hGDNF) is shown in Figure 8C middle.

[0191] Regulatable constructs (Figures 8A and 8B) consisted of a single rAAV vector carrying the activation domain fusion protein and DNA binding domain fusion protein components encoding the hGDNF gene and transcription factors. In both constructs, expression of hGDNF was driven by a minimal IL-2 promoter adjacent to the binding site for eight transcription factors for dimerization, described in more detail below.

[0192] In the construct (pPAAV-TF-Z8-hGDNF) illustrated in Figure 8A, the CMV enhancer / promoter drives the expression of a single transcript enc...

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Abstract

Recombinant adeno-associated viruses (rAAV) for delivery of tunable transgenes to the central nervous system (CNS) of mammals are provided. Also provided are methods of using the vectors to treat neurodegenerative diseases in subjects, kits for constructing or using the vectors or carrying out the methods of the invention. The transgene sequence is expressed from a promoter / enhancer region that contains binding sites for one or more transcription factors that are responsive to small molecule inducers. Both the transgenic construct and the construct comprising the transcription factor are delivered to the target cells. The tunable transgene can be delivered on the same rAAV vector as the transcription factor, or on a different vector. The transcription factor may comprise two polypeptide chains, e.g., a DNA binding domain and a transcriptional activation domain, which form an active dimer in the presence of a dimer such as rapamycin or a non-immunogenic analog thereof . The vectors, methods and kits of the present invention can be used to deliver a gene such as AADC or GDNF to the brain of a patient suffering from a neurodegenerative disease such as Parkinson's disease, wherein the expression of AADC or GDNF in the brain can then be controlled by using Rapa The patient is treated with mycetomycin or its analogs to adjust.

Description

technical field [0001] The present invention relates to the regulation of genes introduced by gene therapy, specifically the regulation of the expression of transgenes transduced into the central nervous system (CNS) of mammals. Background technique [0002] Many human diseases are caused by abnormal expression of genes. When a gene is underexpressed, or when the gene product itself is defective, the absence of the corresponding functional gene product can be treated by delivering the missing gene product to the patient. However, delivery of proteins is often difficult and only confers benefit for a limited period of time, meaning that the protein must be re-administered repeatedly on a regular basis, perhaps requiring occasional dosing, such as in the presence of chronic disease. Repeat dosing is costly, inconvenient, and can suffer from poor patient compliance. Furthermore, the level of a gene product can change significantly between the time immediately after administra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/864A61P25/16A61K48/00A61K35/76
CPCA61K48/00C12N15/86C12N2750/14143C12N2830/00C12N2830/42C12N2830/85C12N2840/203A61P25/00A61P25/16
Inventor L·M·桑夫特内尔V·M·里弗拉
Owner GENZYME CORP
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