Processes for packaging aggregated 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 oligonucleotides comprise at their 5′ end at least 3 and at most 15 guanosine entities and at their 3′ end at least 3 and at most 15 guanosine entities, and wherein said oligonucleotides comprise 20-40 nucleotides, and wherein said oligonucleotides are packaged into said virus-like particle, said process comprising the steps of:

(a) providing coat protein of said RNA bacteriophage;

(b) providing a nucleotide composition comprising said aggregated oligonucleotides, wherein said nucleotide composition is obtainable by a process for producing said nucleotide composition, said process comprising the steps of:

(i) providing said at least two oligonucleotides in solution I; wherein said solution I comprises an alkaline pH;

(ii) disaggregating said oligonucleotides, wherein said disaggregating comprises the steps of

(a) adjusting the temperature of solution I to temperature I, wherein said temperature I is 20 to 70° C.;

(b) incubating said oligonucleotides in said solution I at said temperature I, wherein said incubating is performed until said oligonucleotides comprise a relative peak start time above 110%, wherein said relative peak start time is determined in size exclusion HPLC with the capsid of an RNA bacteriophage as the standard; and

(c) adjusting the temperature of said solution I to temperature II, wherein said temperature II is 0 to 10° C.

(iii) adjusting the pH of said solution I to pH 5 to 8; and

(iv) aggregating said oligonucleotides, wherein said aggregating comprises the steps of:

(a) providing said oligonucleotides in said solution I and generating a solution II, wherein said solution II further comprises at least 20 mM of a cation, wherein said cation is selected from the group consisting of Na + , K + , NH 4 + , Li + , Ca 2+ , and Mg 2+ ;

(b) adjusting the temperature of solution II to temperature III, wherein said temperature III is 50 to 99° C.;

(c) incubating said oligonucleotides in solution II at temperature III, wherein said incubating is performed until said oligonucleotides comprise a relative peak start time of 50 to 110%; wherein said relative peak start time is determined in size exclusion HPLC with the capsid of an RNA bacteriophage as the standard; and

(d) adjusting the temperature of solution II to temperature IV, wherein said temperature IV is 0 to 10° C.;

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

(i) said coat protein;

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

(iii) said aggregated oligonucleotides;

(d) removing said agent from said mixture; and

(e) 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 of a RNA bacteriophage.

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

4. 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 (MX1 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).

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

6. The process of claim 1 , wherein said agent comprises a denaturing compound selected from urea and guanidiniumhydrochloride.

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

8. 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, wherein the concentration of said sodium chloride in said first buffer is 50 to 350 mM.

9. The process of claim 8 , wherein said first buffer exchange is performed across a membrane, wherein said membrane comprises a molecular weight cut off of 5 to 30 kD.

10. The process 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+ .

11. 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.

12. The process of claim 1 , wherein said solution I comprises a pH of 8 to 13.

13. The process of claim 1 , wherein the concentration of said oligonucleotides in said solution II is 50 μM to 2 mM.

14. The process of claim 1 , wherein said oligonucleotides in solution II comprise a relative peak start time of 80 to 110%, wherein said relative peak start time is determined in size exclusion HPLC with the capsid of an RNA bacteriophage as the standard.

15. The process of claim 1 , wherein said oligonucleotides have the nucleic acid sequence “G10” GGGGGGGGGG GACGATCGTC GGGGGGGGGG (SEQ ID NO:8).

16. The process of claim 1 , wherein said coat protein consists of the sequence of SEQ ID NO:10 (Qβ CP).

17. The process of claim 6 , wherein said denaturing compound is urea and wherein the concentration of said urea in said mixture is 0.25 to 7.2 M.

18. The process of claim 1 , wherein said adjusting of the temperature of said solution I to temperature II is performed with a temperature ramp of at least 3.6° C./min.

19. The process of claim 1 , wherein said adjusting the temperature of solution II to temperature IV is performed with a temperature ramp of least 3.6° C./min.

20. The process of claim 1 , wherein said oligonucleotides comprise a palindromic sequence.