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Herpes virus vectors for dendritic cells

a technology of dendritic cells and herpes simplex, which is applied in the field of attenuated herpes simplex viruses, can solve the problems of retroviruses not giving high-efficiency gene delivery to dendritic cells, affecting the antigen processing capabilities of infected cells, and hampered approach, etc., to improve antigen processing capability, and achieve high-efficiency gene delivery

Inactive Publication Date: 2005-11-10
BIOVEX LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0008] These results show that while efficient gene delivery to dendritic cells can be achieved relatively easily using HSV vectors, for the cells to retain their antigen processing capabilities the particular combination of mutations in the virus must be carefully chosen. The invention thus for the first time provides viral vectors for dendritic cells which provide highly efficient gene delivery, without adversely affecting the antigen processing capabilities of the infected dendritic cells.

Problems solved by technology

However effective transfer of antigens into DCs for any of these targets has proved the greatest problem with this approach.
Secondly, following the identification of the antigen / antigens, it is necessary to deliver the antigens in an immunogenic form to the immune system.
However this approach has been hampered by the absence of efficient means by which to load these cells with antigens.
As regard to viral vectors, retroviruses do not give high efficiency gene delivery to dendritic cells (Reeves er al.
1996; Aicher et al., 1997), and in our hands, unlike work reported by others (Arthur et al., 1997), adenoviruses only give low efficiency gene delivery.

Method used

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  • Herpes virus vectors for dendritic cells

Examples

Experimental program
Comparison scheme
Effect test

example 1

Genes can be Efficiently Delivered to Dendritic Cells Using HSV Vectors

[0068] These experiments were aimed to determine if HSV could infect and deliver genes to dendritic cells, using essentially wild type viruses. Multiplication of infection (MOI) of between 0.1 to 10 were used. Here in each case 1×105 dendritic cells were infected by gentle pelleting, resuspension in about 100 μl virus suspension in DMEM, incubation at 37° C. for 1 hr, then transfer into 24 well plates with 2 ml of RPMI / 10% FCS+100 ng / ml GM-CSF, 50 ng / ml IL-4. These plates were then incubated at 37° C. / 5% CO2 for the remainder of the experiment. Viruses 17+pR20.5 / UL43 and 17+ / pR20.5 / US5 were used for these experiments, each mutated for a different gene (UL43 or US5) by the insertion of the pR20.5 cassette. Both UL43 and US5 have been previously identified as being unnecessary for growth of HSV in culture, and as not affecting the kinetics of infection in mouse models in vivo. The efficiency of gene delivery was a...

example 2

Dendritic Cells are Relatively Non-Permissive for the Growth of HSV, Inactivation of UL43 Reducing Growth Further

[0069] To determine whether the efficient gene delivery observed in Example 1 was due to lytic replication of the viruses, growth curves were performed in which dendritic cells were infected at MOIs ranging from 0.1 to 10, as in Example 1, and virus growth quantified over time. At 24 hr intervals samples of the culture media were tired onto BHK cells− permissive for the growth of HSV− and numbers of plaques counted.

[0070] Results

TABLE 2Total virus in dendritic cell culture media (pfu)Time after17+ / pR20.5 / UL4317+ / pR20.5 / US5infectionMOIMOI(days)0.11100.11100100001000001000000100001000010000001200100000300000100100000100002200010000600001000040000800032000010002000030000030000200041000100010000100000200002000

[0071] As shown in Table 2 above, these experiments demonstrated that while a limited productive infection can occur in dendritic cell cultures infected with HSV thi...

example 3

Different HSV Mutants Show Different Levels of Gene Delivery Efficiency and Toxicity in Dendritic Cells

[0072] The virus strains (i)-(x) described above were used to infect dendritic cells at MOI's in a rage between 0.1-10 as in Example 1. At intervals duplicate samples (2×100 μl of the culture) were taken and one sample viewed for GFP expression (fluorescence microscopy) and / or stained with X-gal by gentle pelleting and resuspension in fix (0.811 / 0 glutaraldehyde for 10 min) followed by X-gal buffer (as in Coffin et al, 1996) and incubation at 37° C. for 2 hr, depending on the nature of the insertion in the particular virus under test. The other sample was stained with trypan blue-live cells excluding the stain, and the number of non-staining cells as a percentage of the number of cells in the original culture counted. In each individual set of experiments, which were repeated twice and in which usually three viruses were tested at a time, virus (i) was used as an internal control ...

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Abstract

An attenuated herpes virus capable of efficiently infecting a dendritic cell without preventing antigen processing occurring within the infected cell. The attenuated herpes virus and dendritic cells infected with the virus are useful immunotherapeutic methods of treating disease.

Description

FIELD OF THE INVENTION [0001] The present invention relates to attenuated herpes simplex viruses capable of efficiently infecting dendritic cells. It also relates to the use of such viruses in immunotherapy approaches to the treatment of disease. BACKGROUND TO THE INVENTION [0002] Dendritic cells (DCs) are the most potent antigen presenting cells and are efficient at inducing responses even to antigens to which the immune system has become tolerant. Thus for tumour immunotherapy, in which an immune response is raised against a tumour, the use of DCs may be ideal if they were made to present tumour specific antigens. DCs mist also be used to present antigens derived from infectious agents such as bacteria, viruses or parasites, providing protective or therapeutic vaccines for such diseases. However effective transfer of antigens into DCs for any of these targets has proved the greatest problem with this approach. [0003] To provide a realistic chance of generating a therapeutic immune...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/09A61K35/14A61K35/76A61K39/00A61K39/12A61K48/00A61P31/00A61P35/00A61P35/02A61P37/02C12N5/10C12N7/00C12N7/01C12N7/04C12N15/869C12R1/91C12R1/93
CPCA61K48/00A61K2039/5154C12N2710/16643C12N15/86A61K2039/5254A61P31/00A61P33/00A61P35/00A61P35/02A61P37/00A61P37/02A61K35/76
Inventor COFFIN, ROBERT STUARTCHAIN, BENJAMIN
Owner BIOVEX LTD
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