Construct for epigenetic modification and its use in the silencing of genes
Herein described is a construct for epigenomic modification of genes composed of: (a) a Krüppel-associated box zinc finger protein or homologous, (b) a DNA region capable of binding to the target gene or homologous, (c) a human DNA methyltransferase DNMT3A or homologous and (d) a murine DNA methyltransferase Dnmt3L or homologous, wherein components a), b), c) and d) are linked to each other either directly or via at least one linker. The construct is a designer epigenome modifier that can be used to silence genes coding for a protein in leukocytes that avoids the internalization of HI viruses in immune cells.
1. A single molecule for stably silencing expression of a target gene of interest that is endogenous to a human primary cell via targeted epigenomic modification, said molecule comprising a fusion protein or a nucleic acid encoding said fusion protein, wherein said fusion protein comprises the following components:
a) a Krüppel-associated box (KRAB) effector domain,
b) a DNA binding domain capable of binding to said target gene,
c) a human DNA methyltransferase DNMT3A or a homolog thereof that exhibits at least 90% sequence identity to SEQ ID NO: 3, and
d) a murine DNA methyltransferase Dnmt3L or a homolog thereof that exhibits at least 90% sequence identity to SEQ ID NO: 5,
whereby components a), b), c) and d) are linked to each other either directly or via at least one linker.
2. The molecule according to claim 1 , characterized in that said single molecule is a fusion protein.
3. The molecule according to claim 1 , characterized in that said single molecule is a nucleic acid molecule.
4. A vector comprising the molecule according to claim 3 .
5. The vector according to claim 4 , characterized in that said vector is derived from a lentivirus, an adenovirus or an adeno-associated virus.
6. The molecule according to claim 3 , characterized in that said single molecule is an mRNA molecule that optionally further comprises one or more additional components selected from the group consisting of a 7-methylguanosine cap at the 5′-end, an artificial cap analogue at the 5′-end, a non-coding region at the 5′-end, a non-coding region at the 3′-end and a polyA tail at the 3′-end.
7. The molecule according to claim 3 , characterized in that said nucleic acid molecule is a DNA molecule.
8. The single molecule of claim 1 , wherein said target gene of interest is a human T-cell receptor selected from the group consisting of human immunodeficiency virus (HIV) co-receptors CCR5 and CXCR4.
9. The single molecule of claim 1 , wherein said Krüppel-associated box (KRAB) effector domain is capable of recruiting KRAB-associated protein 1 (KAP 1) that, in turn induces histone modification.
10. The single molecule of claim 1 , wherein said Krüppel-associated box (KRAB) effector domain comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 90% identical to SEQ ID NO: 1.
11. The single molecule of claim 1 , wherein said DNA binding domain capable of binding to said target gene of interest is a transcription-activator-like effector-based DNA binding domain (TALE-DBD) having the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 7.
12. A method for stably silencing expression of a target gene of interest that is endogenous to a human primary cell, said method comprising the steps of:
(a) introducing a single molecule according to claim 1 into human primary cells; and
(b) silencing the gene of interest in said human primary cells via epigenetic modification.
13. The method according to claim 12 , wherein said single molecule is an mRNA molecule.
14. The method according to claim 13 , wherein step (a) is performed ex vivo.
15. The method according to claim 12 , wherein said human primary cells are isolated T lymphocytes, and said target gene of interest is a human T-cell receptor selected from the group consisting of HIV co-receptors CCR5 and CXCR4.