Nucleic acids for reducing carbohydrate epitopes

Details Nucleic acids for reducing carbohydrate epitopes Nucleic acids for reducing carbohydrate epitopes

1999-06-11

2002-06-04

Crouch, Deborah (Department: 1632)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C435S069100, C435S070100, C435S455000, C424S093200, C424S093210

Reexamination Certificate

active

06399758

ABSTRACT:

The present invention relates to nucleic acids which encode glycosyltransferase and are useful in producing cells and organs from one species which may be used for transplantation into a recipient of another species. Specifically the invention concerns production of nucleic acids which, when present in cells of a transplanted organ result in reduced levels of antibody recognition of the transplanted organ.
The transplantation of organs is now possible due to major advances in surgical and other techniques. However, availability of suitable human organs for transplantation is a significant problem. Demand outstrips supply. This has caused researchers to investigate the possibility of using non-human organs for transplantation.
Xenotransplantation is the transplantation of organs from one species to a recipient of a different species. Rejection of the transplant in such cases is a particular problem, especially where the donor species is more distantly related, such as donor organs from pigs and sheep to human recipients. Vascular organs present a special difficulty because of hyperacute rejection (HAR).
HAR occurs when the complement cascade in the recipient is initiated by binding of antibodies to donor endothelial cells.
Previous attempts to prevent HAR have focused on two strategies: modifying the immune system of the host by inhibition of systemic complement formation (
1
,
2
) and antibody depletion (
3
,
4
). Both strategies have been shown to temporarily prolong xenograft survival. However, these methodologies are therapeutically unattractive in that they are clinically impractical and would require chronic immunosuppressive treatments. Therefore, recent efforts to inhibit HAR have focused on genetically modifying the donor xenograft. One such strategy has been to achieve high-level expression of species-restricted human complement inhibitory proteins in vascularized pig organs via transgenic engineering (
5
-
7
). This strategy has proven to be useful in that it has resulted in the prolonged survival of porcine tissues following antibody and serum challenge (
5
,
6
). Although increased survival of the transgenic tissues was observed, long-term graft survival was not achieved (
6
). As observed in these experiments and also with systemic complement depletion, organ failure appears to be related to an acute antibody-dependent vasculitis (
1
,
5
).
In addition to strategies aimed at blocking complement activation on the vascular endothelial cell surface of the xenograft, recent attention has focused on identification of the predominant xenogeneic epitope recognised by high-titre human natural antibodies. It is now accepted that the terminal galactosyl residue, Gal-&agr;(1,3)-Gal, is the dominant xenogeneic epitope (
8
-
15
). This epitope is absent in Old World primates and humans because the &agr;(1,3)-galactosyltransferase (gal-transferase or GT) is non-functional in these species. DNA sequence comparison of the human gene to &agr;(1,3)-galactosyltransferase genes from the mouse (
16
,
17
), ox (
18
), and pig (
12
) has revealed that the human gene contained two frameshift mutations, resulting in a non-functional pseudogene (
20
,
21
). Consequently, humans and Old World primates have pre-existing high-titre antibodies directed at this Gal-&agr;(1,3)-Gal moiety as the dominant xenogeneic epitope.
It appears that different glycosyltransferases can compete for the same substrate. Hence &agr;(1,2)-fucosyltransferase or H transferase (HT) (
22
) could be an appropriate enzyme to decrease the expression of Gal-&agr;(1,3)-Gal, as both the &agr;(1,2)-fucosyltransferase and the &agr;(1,3)-galactosyltransferase use N-acetyl lactosamine as an acceptor substrate, transferring fucose or galactose to generate fucosylated N-acetyl lactosamine (H substance) or Gal-&agr;(1,3)-Gal, respectively. Furthermore, the &agr;(1,3)-galactosyltransferase of most animals cannot use the fucosylated N-acetyl lactosamine as an acceptor to transfer the terminal galactose, but will only transfer to N-acetyl lactosamine residues (
23
). We have previously reported that the simultaneous expression of two glycosyltransferases, &agr;(1,2)-fucosyltransferase (H transferase) and &agr;(1,3)-galactosyltransferase, does not lead to equal synthesis of each monosaccharide, but the activity of the &agr;(1,2)-fucosyltransferase is given preference over that of the &agr;(1,3)-galactosyltransferase, so that the expression of Gal-&agr;(1,3)-Gal is almost entirely suppressed (
24
).
The &agr;(1,3)-galactosyltransferase (Gal transferase) can galactosylate two types of precursor chains: Type 1: Gal&bgr;(1,3)GlcNAc and Type 2: Gal&bgr;(1,4)GlcNAc.
Furthermore, both of these precursors can be transformed into H substance or fucosylated &bgr;-D-Gal by two &agr;(1,2)-fucosyltransferases (
25
,
26
). These two fucosyltransferases are H-transferase or FUT1 (
22
) and secretor (Se) transferase or FUT2 (
27
). While both enzymes can use both types of precursors, FUT1 HT preferentially utilises Type 2 precursor chains, and FUT2 preferentially utilises Type 1 (
28
).
In work leading up to the present invention the inventors set out to create a nucleic acid which would be useful in reducing unwanted carbohydrate epitopes on the surface of cells. The nucleic acid could be used in production of an organ which would cause reduced levels of rejection when transplanted into another species. The inventors surprisingly found that a glycosyltransferase derived from porcine origin was useful in decreasing unwanted carbohydrate epitopes in cells. The enzyme encoded by the nucleic acid is able to compete effectively with glycosyltransferases which produce unwanted carbohydrate epitopes. In this particular work the inventors cloned a secretor transferase (Se) gene from pig origin, and demonstrated that this is expressed in cells and results in reduced levels of unwanted epitopes on those cells. The secretor transferase is referred to herein as “pig secretor”.
SUMMARY OF THE INVENTION
In a first aspect the invention provides a nucleic acid encoding a first glycosyltransferase which is able to compete with a second glycosyltransferase for a substrate when said nucleic acid is expressed in a cell which produces said second glycosyltransferase, resulting in reduced levels of a product from said second glycosyltransferase.
The nucleic acid may be DNA or RNA, single or double stranded, or covalently closed circular. It will be understood that the nucleic acid encodes a functional gene (or part thereof) which enables a glycosyltransferase with the appropriate activity to be produced. Preferably the nucleic acid is in an isolated form; this means that the nucleic acid is at least partly purified from other nucleic acids or proteins.
Preferably the nucleic acid comprises the correct sequences for expression, more preferably for expression in a eukaryotic cell. The nucleic acid may be present on any suitable vehicle, for example, a eukaryotic expression vector such as pcDNA (Invitrogen). The nucleic acid may also be present on other vehicles, whether suitable for eukaryotes or not, such as plasmids, phages and the like.
Preferably the first glycosyltransferase is a an enzyme with a higher affinity for the substrate than said second glycosyltransferase. More preferably said first glycosyltransferase preferentially utilises Type 1 substrates. Still more preferably said first glycosyltransferase is Se (also known as FUT2). Preferably the Se originates or is derived from, or is based on, Se from the same species as the cell in which it is intended to be expressed. Thus, the first glycosyltransferase and the cell in which the enzyme is expressed may each originate from animals of the same species. Such species may be pig, New World monkey, dog or other suitable species. The nucleic acid encoding Se is not necessarily directly derived from the native gene. The nucleic acid sequence for Se may be made by PCR, constructed de novo or cloned.
More preferably Se is of porcine origin or based on the porcine enzyme. This means that the enzyme is based on, homologo

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