Packaging oligonucleotides into virus-like particles
Inventors
WALTERS, EVAN DAVID • Hennecke, Frank
Assignees
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Publication Number
US-12084655-B2
Publication Date
2024-09-10
Expiration Date
Abstract
The present invention relates to processes for producing compositions comprising (i) a virus-like particle of an RNA bacteriophage, and (ii) aggregated oligonucleotides, wherein said aggregated oligonucleotides are packaged into said virus-like particle. The invention further provides processes for producing nucleotide compositions comprising aggregated oligonucleotides suitable for use in the aforementioned processes before. Moreover, the invention further provides nucleotide compositions comprising aggregated oligonucleotides. Furthermore, the invention further provides compositions comprising (i) a virus-like particle of an RNA bacteriophage, and (ii) aggregated oligonucleotides, wherein said aggregated oligonucleotides are packaged into said virus-like particle.
Core Innovation
The invention relates to a process for producing a nucleotide composition comprising aggregated oligonucleotides. The process provides oligonucleotides comprising 3 to 15 guanosine entities at the 5′ end and 3 to 15 guanosine entities at the 3′ end, wherein the oligonucleotides comprise phosphodiester connected deoxynucleotides. The process includes denaturing and aggregating steps designed to form aggregates having a target average diameter measured by Dynamic Light Scattering (DLS).
In the denaturing step, an aqueous solution I comprising the oligonucleotides and a chaotropic agent is incubated at 75°C to 99°C until the average diameter of the oligonucleotides is 1 nm or less, as determined by DLS. The aggregating step then incubates an aqueous solution II comprising the oligonucleotides having said average diameter of 1 nm or less, a chaotropic agent, and a cation at 75°C to 99°C to form aggregated oligonucleotides until the average diameter of the formed aggregated oligonucleotides is 7-14 nm, as determined by DLS, followed by adjusting the temperature of solution II to below 40°C.
The described approach addresses prior processes by targeting control of size and conformation using DLS-based endpoints and by using a chaotrope/denaturing-aggregation approach rather than relying on prior disaggregation/aggregation windows. The document contrasts prior outcomes with variable DLS size/conformation, reduced VLP purity and stability, and malformed rod-like aggregates versus exclusively spherical VLPs for the inventive process. It also states that the improved approach enables a wider aggregation operating window and yields improved VLP yield, purity, and stability, and includes the ability to store denatured solutions with low re-aggregation risk.
Claims Coverage
The independent claim is clm-00001. The inventive coverage centers on denaturing guanosine-rich, phosphodiester-connected deoxynucleotide oligonucleotides in a chaotropic aqueous solution to a DLS average diameter of 1 nm or less, followed by aggregating in a chaotropic and cation-containing aqueous solution to a DLS average diameter of 7-14 nm, then cooling to below 40°C. The claim set includes multiple dependent refinements, including restricted chaotropic agent selections, constraints on ionic conditions, HPLC purity criteria, and DLS diameter endpoint narrowing.
DLS-controlled denaturation in chaotropic aqueous solution
Denaturing oligonucleotides by incubating an aqueous solution I comprising said oligonucleotides and a chaotropic agent at 75°C to 99°C until the average diameter of said oligonucleotides is 1 nm or less, wherein said average diameter is determined by Dynamic Light Scattering (DLS).
Chaotropic and cation aggregation to a DLS-defined 7-14 nm endpoint
Aggregating oligonucleotides by incubating an aqueous solution II comprising said oligonucleotides having said average diameter of 1 nm or less obtained in step (b), a chaotropic agent and a cation at 75°C to 99°C to form said aggregated oligonucleotides, wherein said incubating is performed until the average diameter of said formed aggregated oligonucleotides is 7-14 nm, wherein said average diameter is determined by Dynamic Light Scattering (DLS).
Cooling aggregated oligonucleotides below 40°C after DLS-defined aggregation
Adjusting the temperature of said solution II to below 40°C after aggregating to the DLS-defined average diameter of 7-14 nm.
Restricting oligonucleotides to 3-15 guanosine entities at both ends with phosphodiester-connected deoxynucleotides
Providing oligonucleotides comprising 3 to 15 guanosine entities at the 5′ end and 3 to 15 guanosine entities at the 3′ end, wherein said oligonucleotides comprise phosphodiester connected deoxynucleotides.
Avoiding spontaneous self-aggregation by limiting mono- or divalent ions in aqueous solution I
Executing the process such that the aqueous solution I does not contain mono- or divalent ions at concentrations that would cause the oligonucleotides to spontaneously self-aggregate.
Selecting a defined set of chaotropic agents for denaturation
The chaotropic agent in the solution is selected from urea, phenol, isopropyl alcohol, ethanol, and guanidinium chloride.
Producing oligonucleotides with HPLC purity of 90% or higher
Producing oligonucleotides with a purity of 90% or higher, as determined by HPLC.
Using a narrower DLS aggregated-diameter incubation endpoint of 9-14 nm
Including incubating aggregated oligonucleotides until their average diameter is 9-14 nm, with the diameter measured by Dynamic Light Scattering (DLS).
Employing specific guanosine-rich nucleic acid sequence embodiments
The oligonucleotides include one of the nucleic acid sequences listed as G10, G10-11, G12-11, G6, G7, G8, G9, G11, G6-10, G7-10, G8-10, or G9-10 with their corresponding SEQ ID numbers.
Across the independent claim and dependent refinements, the claimed invention is directed to forming aggregated oligonucleotides with DLS-defined size endpoints by first denaturing guanosine-rich phosphodiester-connected deoxynucleotide oligonucleotides in a chaotropic aqueous solution to 1 nm or less, then aggregating in a chaotropic and cation-containing aqueous solution to 7-14 nm, followed by cooling below 40°C, with additional claim refinements controlling chaotropic agent identity, ionic conditions, DLS endpoint, and HPLC purity.
Stated Advantages
Improved process performance versus prior approaches, including higher VLP yield and improved VLP purity.
Improved VLP stability.
Control of size and conformation using DLS-defined endpoints, independent of oligonucleotide purity.
Wider aggregation operating window for GMP scale.
Ability to store denatured solutions with low re-aggregation risk.
Documented Applications
Production of virus-like particles (VLPs) of RNA bacteriophages using aggregated polyG-rich oligonucleotides, including described outcomes contrasting prior processes and showing exclusively spherical VLPs for the inventive process.
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