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Gene delivery vectors provided with a tissue tropism for smooth muscle cells, and/or endothelial cells

a tissue tropism and gene technology, applied in the field of biotechnology, can solve the problems of insufficient in vivo delivery capacity of adenovirus vectors, limited use of current vectors in specific applications, and inability to easily transduce endothelial cells and smooth muscle cells

Inactive Publication Date: 2005-08-04
JANSSEN VACCINES & PREVENTION BV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0026] The present invention provides gene therapy methods, compounds and medicines. The present invention is particularly useful in gene therapy applications where endothelial cells and / or smooth muscle cells form the target cell type. The present invention relates to gene delivery vehicles provided with a tissue tropism for at least endothelial cells and / or smooth muscle cells. The present invention further relates to gene delivery vehicles having been deprived of a tissue tropism for liver cells.

Problems solved by technology

However, some characteristics of the current vectors limit their use in specific applications.
For instance, endothelial cells and smooth muscle cells are not easily transduced by the current generation of adenovirus vectors.
On the other hand, in some applications, even the very good in vivo delivery capacity of adenovirus vectors is not sufficient and higher transfer efficiencies are required.
However, the fact that (at least some) members of these subgroups are able to bind to CAR does not exclude that these viruses have different infection efficiencies in various cell types.
However, there is still a number of drawbacks associated with the use of adenoviral vectors: 1) Adenoviruses, especially the well investigated serotypes Ad2 and Ad5, usually elicit an immune response in the host into which they are introduced; 2) it is currently not feasible to target the virus to certain cells and tissues; 3) the replication and other functions of the adenovirus are not always very well suited for the cells; which are to be provided with the additional genetic material; and 4) the serotypes Ad2 or Ad5 are not ideally suited for delivering additional genetic material to organs other than the liver.
Although these methods mostly succeed in avoiding gross delivery of the vector to the liver, most of the methods are crude and still have considerable leakage and / or have poor target tissue penetration characteristics.
In some cases, inadvertent delivery of the vector to liver cells can be toxic to the patient.
For instance, delivery of a herpes simplex virus (HSV) thymidine kinase (TK) gene for the subsequent killing of dividing cancer cells through administration of gancyclovir is quite dangerous when also a significant amount of liver cells are transduced by the vector.
Significant delivery and subsequent expression of the HSV-TK gene to liver cells is associated with severe toxicity.

Method used

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  • Gene delivery vectors provided with a tissue tropism for smooth muscle cells, and/or endothelial cells
  • Gene delivery vectors provided with a tissue tropism for smooth muscle cells, and/or endothelial cells
  • Gene delivery vectors provided with a tissue tropism for smooth muscle cells, and/or endothelial cells

Examples

Experimental program
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Effect test

example 1

Generation of Adenovirus Serotype 5 Based Viruses with Chimeric Fiber Proteins

Generation of Adenovirus Template Clones Lacking DNA Encoding for Fiber

[0094] The fiber coding sequence of adenovirus serotype 5 is located between nucleotides 31042 and 32787. To remove the adenovirus serotype 5 DNA encoding fiber we started with construct pBr / Ad.Bam-rITR (FIG. 1; ECACC deposit P97082122). From this construct a NdeI site was first removed. For this purpose, pBr322 plasmid DNA was digested with NdeI after which protruding ends were filled using Klenow enzyme. This pBr322 plasmid was then re-ligated, digested with NdeI and transformed into E. coli DH5α. The obtained pBr / ΔNdeI plasmid was digested with ScaI and SalI and the resulting 3198 bp vector fragment was ligated to the 15349 bp ScaI-SalI fragment derived from pBr / Ad.Bam-rITR, resulting in plasmid pBr / Ad.Bam-rITRΔNdeI which hence contained a unique NdeI site. Next a PCR was performed with oligonucleotides “NY-up” and “NY-down” (FIG....

example 2

Biodistribution of Chimeric Viruses after Intravenous Tail Vein Injection of Rats

[0099] To investigate the biodistribution of the chimeric adenoviruses carrying fiber 12, 16, 28, or 40-2, 1×1010 particles of each of the generated virus batches was diluted in 1 ml PBS after which the virus was injected in the tail vein of adult male Wag / Rij rats (3 rats / virus). As a control, Ad5 carrying the luciferase transgene was used. Forty-eight hours after the administration of the virus, the rats were sacrificed after which the liver, spleen, lung, kidney, heart, and brain were dissected. These organs were subsequently mixed with 1 ml of lysis buffer (1% Triton X-100 / PBS) and minced for 30 seconds to obtain a protein lysate. The protein lysate was subsequently tested for the presence of transgene expression (luciferase activity) and the protein concentration was determined to express the luciferase activity per μg of protein. The results, Shown in Table II, demonstrate that in contrast to the...

example 3

Chimeric Viruses Display Differences in Endothelial and Smooth Muscle Cell Transduction

A) Infection of Human Endothelial Cells

[0100] Human endothelial cells (HUVEC) were isolated, cultured and characterized as described previously (Jaffe et al. 1973; Wijnberg et al. 1997). Briefly, cells were cultured on gelatin-coated dishes in M199 supplemented with 20 mM HEPES, pH 7.3 (Flow Lab., Irvine, Scotland), 10% (v / v) human serum (local blood bank), 10% (v / v) heat-inactivated newborn calf serum (NBCS) (GIBCO BRL, Gaithersburg, Md.), 150 μg / ml crude endothelial cell growth factor, 5 U / ml heparin (Leo Pharmaceutics Products, Weesp, The Netherlands), penicillin (100 IU / ml) / streptomycin (100 μg / ml) Boehringer Mannheim, Mannheim, Del.) at 37° C. under 5% (v / v) CO2 / 95% (v / v) air atmosphere. Cells used for experiments were between passage 1-3. In a first set of experiments 40000 HUVEC cells (a pool from 4 different individuals) were seeded in each well of 24-well plates in a total volume of 20...

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Abstract

A gene delivery vehicle having been provided with at least a tissue tropism for cells selected from the group of smooth muscle cells, endothelial cells, and / or liver cells. The tissue tropism is generally provided by a virus capsid, such as one comprising protein fragments from at least two different viruses, such as two different adenoviruses, including adenovirus of subgroup C or subgroup B (for example, adenovirus 16). The protein fragments can comprise a tissue tropism-determining fragment of a fiber protein derived from a subgroup B adenovirus. Also, cells for producing such gene delivery vehicles and pharmaceutical compositions containing these gene delivery vehicles are provided. Further, a method is disclosed for delivering nucleic acid to cells such as smooth muscle cells and / or endothelial cells which involves administering to the cells an adenovirus capsid having proteins from at least two different adenoviruses and wherein at least a tissue tropism-determining fragment of a fiber protein is derived from a subgroup B adenovirus. Particular constructs are also disclosed.

Description

CROSS-REFERENCE TO RELATED APPLICATION [0001] This application is a continuation of co-pending application Ser. No. 09 / 444,284, filed Nov. 19, 1999, now U.S. Pat. No. ______, which is a continuation-in-part of application Ser. No. 09 / 348,354, filed Jul. 7, 1999, abandoned, the contents of both of which are incorporated by this reference.TECHNICAL FIELD [0002] The invention relates generally to biotechnology and, more particularly, to the field of molecular genetics and medicine. In particular, the present invention relates to the field of gene therapy and, more particularly, to gene therapy using adenoviruses. BACKGROUND [0003] In gene therapy, genetic information is usually delivered to a host cell in order to either correct (supplement) a genetic deficiency in the host cell, or to inhibit an undesired function in the host cell, or to eliminate the host cell. Of course, the genetic information can also be intended to provide the host cell with a desired function, e.g., to supply a ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/00A61K48/00C07K14/075C12N15/861
CPCA61K38/00A61K48/00C07K14/005C07K2319/00C12N2810/6018C12N2710/10322C12N2710/10343C12N2710/10345C12N15/86
Inventor VOGELS, RONALDHAVENGA, MENZO J. E.BOUT, ABRAHAM
Owner JANSSEN VACCINES & PREVENTION BV
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