Processes for packaging oligonucleotides into virus-like particles of RNA bacteriophages

1. A process for producing a composition comprising (i) a virus-like particle, wherein said virus-like particle is a virus-like particle of an RNA bacteriophage, and (ii) aggregated oligonucleotides, wherein said aggregated oligonucleotides are packaged into said virus-like particle, said process comprising the steps of:

(a) generating a mixture, wherein said mixture comprises:

(i) a coat protein of said RNA bacteriophage;

(ii) an agent capable of preventing the self-assembly of said coat protein; and

(iii) aggregated oligonucleotides, wherein said aggregated oligonucleotides comprise oligonucleotides comprising at least one poly G stretch, and wherein said aggregated oligonucleotides comprise a relative peak start time of 50 to 110%, where said relative peak start time is determined by size exclusion HPLC with the capsid of the RNA bacteriophage as the standard;

(b) removing said agent from said mixture; and

(c) allowing said coat protein to self-assemble into a virus-like particle.

2. The process of claim 1 , wherein said coat protein comprises recombinant proteins, or fragments thereof that are capable of self-assembly, of a RNA bacteriophage.

3. The process of claim 1 , wherein said RNA bacteriophage is Qβ.

4. The process of claim 1 , wherein said RNA bacteriophage is AP205.

5. The process of claim 1 , wherein said coat protein comprises a sequence selected from the group consisting of:

(a) SEQ ID NO: 10 (Qβ CP);

(b) a mixture of SEQ ID NO:10 and SEQ ID NO:11 (Qβ A1 protein);

(c) SEQ ID NO: 12 (R17 coat protein);

(d) SEQ ID NO: 13 (fr coat protein);

(e) SEQ ID NO:14 (GA coat protein);

(f) SEQ ID NO: 15 (SP coat protein);

(g) a mixture of SEQ ID NO:15 and SEQ ID NO:16;

(h) SEQ ID NO: 17 (MS2 coat protein);

(i) SEQ ID NO: 18 (M11 coat protein);

(j) SEQ ID NO: 19 (MXI coat protein);

(k) SEQ ID NO:20 (NL95 coat protein);

(l) SEQ ID NO:21 (f2 coat protein);

(m) SEQ ID NO:22 (PP7 coat protein); and

(n) SEQ ID NO:23 (AP205 coat protein).

6. The process of claim 1 , wherein the concentration of said oligonucleotides in said mixture is 25 to 100 μM.

7. The process of claim 1 , wherein the molar ratio of said oligonucleotides and said coat protein in said mixture is 0.5 to 1.2.

8. The process of claim 1 , wherein said agent comprises a denaturing compound selected from urea and guanidinium hydrochloride.

9. The process of claim 1 , wherein said agent further comprises a reducing agent.

10. The process of claim 1 , wherein said removing of said agent from said mixture is performed by a first buffer exchange with a first buffer, wherein said first buffer comprises sodium chloride.

11. The process of claim 10 , wherein said first buffer exchange is performed across a membrane, wherein said membrane comprises a molecular weight cut off of 1 to 50 kD.

12. The process of claim 1 , wherein said process further comprises the step of contacting said virus-like particle with an oxidizing agent, wherein said oxidizing agent is selected from the group consisting of:

(a) hydrogen peroxide;

(b) oxygen;

(c) gluthathion;

(d) Cu 2+ ; and

(e) Fe 3+ .

13. The process of claim 1 , wherein said process further comprises the step of purifying said virus-like particle, and wherein said purifying comprises a second buffer exchange with a second buffer, wherein said second buffer is a pharmaceutically acceptable buffer.

14. The process of claim 13 , wherein said second buffer exchange is performed using a membrane, wherein said membrane comprises a molecular weight cut off of 100 to 1000 kD.

15. The process of claim 1 , wherein the coat protein yield is at least 75% and/or wherein the oligonucleotide yield is at least 75%.