Nucleic acids encoding repetitive amino acid sequences rich in proline and alanine residues that have low repetitive nucleotide sequences

Patent metadata

Field	Value
Publication number	US-11401305-B2
Application number	US-201616064951-A
Country	US
Kind code	B2
Filing date	Dec 22, 2016
Priority date	Dec 22, 2015
Publication date	Aug 2, 2022
Grant date	Aug 2, 2022

Abstract

The present invention relates to a nucleic acid molecule comprising a low repetitive nucleotide sequence encoding a proline/alanine-rich amino acid repeat sequence. The encoded polypeptide comprises a repetitive amino acid sequence that forms a random coil. The nucleic acid molecule comprising said low repetitive nucleotide sequences can further comprise a nucleotide sequence encoding a biologically or pharmacologically active protein. Further, the present invention provides for selection means and methods to identify said nucleic acid molecule comprising said low repetitive nucleotide sequence. The present invention also relates to a method for preparing said nucleic acid molecules. Also provided herein are methods for preparing the encoded polypeptide or drug conjugates with the encoded polypeptide using the herein provided nucleic acid molecules. The drug conjugate may comprise a biologically or pharmacologically active protein or a small molecule drug. Also provided herein are vectors and hosts comprising such nucleic acid molecules.

First claim

The invention claimed is:

1. A nucleic acid molecule, comprising a nucleotide sequence encoding a polypeptide consisting of proline and alanine or a polypeptide consisting of proline, alanine, and serine,
wherein the nucleotide sequence of said nucleic acid has a length of at least 300 nucleotides,
wherein said nucleotide sequence has a Nucleotide Repeat Score (NRS) lower than 1,000,
wherein said Nucleotide Repeat Score (NRS) is determined according to the formula:

NRS
=

∑

n
=
4

N
tot

-
1

⁢

n
2

⁢

∑

i
=
1

k
⁡

(
n
)

⁢

f
i

⁡

(
n
)

N
tot

,

wherein
N tot is the length of said nucleotide sequence,
n is the length of a repeat within said nucleotide sequence, and
f i (n) is the frequency of said repeat of length n,
wherein if there is more than one repeat of length n, k(n) is the number of different repeats of length n, otherwise k(n) is 1 for said repeat of length n.

2. The nucleic acid molecule of claim 1 , wherein said encoded polypeptide consists of proline and alanine.

3. The nucleic acid molecule of claim 2 , wherein proline constitutes more than about 10% and less than about 75% of said encoded polypeptide.

4. The nucleic acid molecule of claim 1 , wherein said encoded polypeptide consists of proline, alanine, and serine.

5. The nucleic acid molecule of claim 4 , wherein proline constitutes more than 4% and less than 40% of said encoded polypeptide.

6. The nucleic acid molecule of claim 1 , wherein said Nucleotide Repeat Score (NRS) is lower than 100.

7. The nucleic acid molecule of claim 1 , wherein said Nucleotide Repeat Score (NRS) is lower than 50.

8. The nucleic acid molecule of claim 1 , wherein said Nucleotide Repeat Score (NRS) is lower than 35.

9. The nucleic acid molecule of claim 1 , wherein the nucleotide sequence of said nucleic acid has a length of at least 900 nucleotides.

10. The nucleic acid molecule of claim 1 , wherein said nucleotide sequence comprises said repeats, wherein said repeats have a maximum length n max , wherein n max is determined according to the formula:

n
max

≤

17
+

N
tot

600

and wherein N tot is the length of said nucleotide sequence.

11. The nucleic acid molecule of claim 1 , wherein said repeats have a maximum length of about 14, 15, 16, or 17 nucleotides to about 55 nucleotides.

12. The nucleic acid molecule of claim 1 , wherein said encoded polypeptide comprises a repetitive amino acid sequence with a plurality of amino acid repeats, wherein no more than 9 consecutive amino acid residues are identical and wherein said polypeptide forms a random coil.

13. The nucleic acid molecule of claim 1 , wherein said nucleic acid molecule is selected from the group consisting of:
(a) a nucleic acid molecule comprising at least one nucleotide sequence selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 192 and SEQ ID NO: 193;
(b) a nucleic acid molecule comprising the nucleotide sequence consisting of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, or SEQ ID NO: 173;
(c) a nucleic acid molecule that hybridizes under stringent conditions to the complementary strand of a nucleotide sequence as defined in (a) or (b);
(d) a nucleic acid molecule comprising a nucleotide sequence having at least 66.7% identity to a nucleotide sequence as defined in any one of (a), (b) and (c); and
(e) a nucleic acid molecule being degenerate as a result of the genetic code to a nucleotide sequence as defined in (a) or (b).

14. The nucleic add molecule of claim 1 , wherein said nucleic add molecule k selected from the group consisting of:
(a) a nucleic add molecule comprising at least one nucleotide sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 194 and SEQ ID NO:

Assignees

• Xl Protein Gmbh
• Univ Muenchen Tech

Inventors

• Binder Uli
• Achatz Stefan
• Skerra Arne

Classifications

• C07K14/5759

  Products of obesity genes, e.g. leptin, obese (OB), tub, fat · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title

• C07K14/00

  Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• C07K14/001 Primary

  by chemical synthesis · CPC title