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JP-7857356-B2 - Liposomes containing lipids with pKa values favorable for RNA delivery

JP7857356B2JP 7857356 B2JP7857356 B2JP 7857356B2JP-7857356-B2

Inventors

  • ギール， アンドリュー

Assignees

  • グラクソスミスクライン バイオロジカルズ ソシエテ アノニム

Dates

Publication Date
20260512
Application Date
20240730
Priority Date
20100706

Claims (20)

  1. A composition comprising liposomes and RNA molecules encoding immunogens, The immunogen includes respiratory syncytial virus (RSV) immunogen, coronavirus spike polypeptide immunogen, or influenza A virus immunogen. The RNA molecule encoding the immunogen includes a polyA tail, The liposome has a lipid bilayer, and the lipid bilayer encapsulates at least half of the RNA molecule encoding the immunogen within an aqueous core. The aforementioned lipid bilayer contains lipids including tertiary amines, The lipid containing the tertiary amine has a pKa in the range of 5.8 to 7.0. The pKa of the lipid containing the tertiary amine in the range of 5.8 to 7.0 is 1) Mix 400 μL of 2 mM of the lipid in 100% by volume ethanol with 800 μL of 0.3 mM fluorescent probe toluenenitrosulfonic acid (TNS) in 90% by volume ethanol and 10% by volume methanol to obtain a lipid/TNS mixture; 2) Mix 7.5 μL of the lipid/TNS mixture obtained in 1) with 242.5 μL of a first buffer, which is a sodium salt buffer containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, and is adjusted to have a pH of 4.44 to 4.52, thereby obtaining a first lipid/buffer mixture; dispense 100 μL of the first lipid/buffer mixture into the first well of a 96-well plate having a clear bottom; 3) Mix 7.5 μL of the lipid/TNS mixture obtained in 1) with 242.5 μL of a second buffer, which is a sodium salt buffer containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, and is adjusted to have a pH of 5.27, thereby obtaining a second lipid/buffer mixture; dispense 100 μL of the second lipid/buffer mixture into the second well of the 96-well plate; 4) Mix 7.5 μL of the lipid/TNS mixture obtained in 1) with 242.5 μL of a third buffer, which is a sodium salt buffer containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, and is adjusted to have a pH of 6.15 to 6.21, thereby obtaining a third lipid/buffer mixture, and dispense 100 μL of the third lipid/buffer mixture into the third well of the 96-well plate; 5) Mix 7.5 μL of the lipid/TNS mixture obtained in 1) with 242.5 μL of a fourth buffer, which is a sodium salt buffer containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, and is adjusted to have a pH of 6.57, thereby obtaining a fourth lipid/buffer mixture; dispense 100 μL of the fourth lipid/buffer mixture into the fourth well of the 96-well plate; 6) Mix 7.5 μL of the lipid/TNS mixture obtained in 1) with 242.5 μL of a fifth buffer, which is a sodium salt buffer containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, and is adjusted to have a pH of 7.10 to 7.20, thereby obtaining a fifth lipid/buffer mixture, and dispense 100 μL of the fifth lipid/buffer mixture into the fifth well of the 96-well plate; 7) Mix 7.5 μL of the lipid/TNS mixture obtained in 1) with 242.5 μL of a sixth buffer, which is a sodium salt buffer containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, and is adjusted to have a pH of 7.72 to 7.80, thereby obtaining a sixth lipid/buffer mixture, and dispense 100 μL of the sixth lipid/buffer mixture into the sixth well of the 96-well plate; 8) Mix 7.5 μL of the lipid/TNS mixture obtained in 1) with 242.5 μL of a seventh buffer, which is a sodium salt buffer containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, and is adjusted to have a pH of 8.27 to 8.33, thereby obtaining a seventh lipid/buffer mixture, and dispense 100 μL of the seventh lipid/buffer mixture into the seventh well of the 96-well plate; 9) Mix 7.5 μL of the lipid/TNS mixture obtained in 1) with 242.5 μL of an eighth buffer, which is a sodium salt buffer containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, and is adjusted to have a pH of 10.47 to 11.12, thereby obtaining an eighth lipid/buffer mixture, and dispense 100 μL of the eighth lipid/buffer mixture into the eighth well of the 96-well plate; 10) Using an excitation wavelength of 322 nm and cutting off wavelengths below 420 nm, the absolute fluorescence of each of the first to eighth wells and the empty well of the 96-well plate at a wavelength of 431 nm was measured. 11) Subtract the absolute fluorescence of the empty well from the absolute fluorescence of each of the first to eighth wells, thereby obtaining the blank removal fluorescence for each of the first to eighth lipid/buffer mixtures; 12) The blank removal fluorescence of each of the first to eighth lipid/buffer mixtures is normalized relative to the blank removal fluorescence of the first lipid/buffer mixture, thereby obtaining a relative fluorescence for each of the first to eighth lipid/buffer mixtures, with the relative fluorescence of the first lipid/buffer mixture set to 1; 13) Plot the relative fluorescence of the first to eighth lipid/buffer mixtures against the pH of the first to eighth lipid/buffer mixtures; 14) The pH corresponding to the relative fluorescence of 0.5 is found from the graph obtained in 13), and the pH corresponding to the relative fluorescence of 0.5 is determined to be the pKa of the lipid. composition.
  2. A composition comprising liposomes and self-replicating RNA molecules encoding immunogens, The immunogen includes respiratory syncytial virus (RSV) immunogen, coronavirus spike polypeptide immunogen, or influenza A virus immunogen. The self-replicating RNA molecule encoding the immunogen includes a poly-A tail, The liposome has a lipid bilayer, and the lipid bilayer encapsulates at least half of the self-replicating RNA molecules encoding the immunogen within an aqueous core. The aforementioned lipid bilayer contains lipids including tertiary amines, The lipid containing the tertiary amine has a pKa in the range of 5.0 to 7.6. The pKa of the lipid containing the tertiary amine in the range of 5.0 to 7.6 is 1) Mix 400 μL of 2 mM of the lipid in 100% by volume ethanol with 800 μL of 0.3 mM fluorescent probe toluenenitrosulfonic acid (TNS) in 90% by volume ethanol and 10% by volume methanol to obtain a lipid/TNS mixture; 2) Mix 7.5 μL of the lipid/TNS mixture obtained in 1) with 242.5 μL of a first buffer, which is a sodium salt buffer containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, and is adjusted to have a pH of 4.44 to 4.52, thereby obtaining a first lipid/buffer mixture; dispense 100 μL of the first lipid/buffer mixture into the first well of a 96-well plate having a clear bottom; 3) Mix 7.5 μL of the lipid/TNS mixture obtained in 1) with 242.5 μL of a second buffer, which is a sodium salt buffer containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, and is adjusted to have a pH of 5.27, thereby obtaining a second lipid/buffer mixture; dispense 100 μL of the second lipid/buffer mixture into the second well of the 96-well plate; 4) Mix 7.5 μL of the lipid/TNS mixture obtained in 1) with 242.5 μL of a third buffer, which is a sodium salt buffer containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, and is adjusted to have a pH of 6.15 to 6.21, thereby obtaining a third lipid/buffer mixture, and dispense 100 μL of the third lipid/buffer mixture into the third well of the 96-well plate; 5) Mix 7.5 μL of the lipid/TNS mixture obtained in 1) with 242.5 μL of a fourth buffer, which is a sodium salt buffer containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, and is adjusted to have a pH of 6.57, thereby obtaining a fourth lipid/buffer mixture; dispense 100 μL of the fourth lipid/buffer mixture into the fourth well of the 96-well plate; 6) Mix 7.5 μL of the lipid/TNS mixture obtained in 1) with 242.5 μL of a fifth buffer, which is a sodium salt buffer containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, and is adjusted to have a pH of 7.10 to 7.20, thereby obtaining a fifth lipid/buffer mixture, and dispense 100 μL of the fifth lipid/buffer mixture into the fifth well of the 96-well plate; 7) Mix 7.5 μL of the lipid/TNS mixture obtained in 1) with 242.5 μL of a sixth buffer, which is a sodium salt buffer containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, and is adjusted to have a pH of 7.72 to 7.80, thereby obtaining a sixth lipid/buffer mixture, and dispense 100 μL of the sixth lipid/buffer mixture into the sixth well of the 96-well plate; 8) Mix 7.5 μL of the lipid/TNS mixture obtained in 1) with 242.5 μL of a seventh buffer, which is a sodium salt buffer containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, and is adjusted to have a pH of 8.27 to 8.33, thereby obtaining a seventh lipid/buffer mixture, and dispense 100 μL of the seventh lipid/buffer mixture into the seventh well of the 96-well plate; 9) Mix 7.5 μL of the lipid/TNS mixture obtained in 1) with 242.5 μL of an eighth buffer, which is a sodium salt buffer containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, and is adjusted to have a pH of 10.47 to 11.12, thereby obtaining an eighth lipid/buffer mixture, and dispense 100 μL of the eighth lipid/buffer mixture into the eighth well of the 96-well plate; 10) Using an excitation wavelength of 322 nm and cutting off wavelengths below 420 nm, the absolute fluorescence of each of the first to eighth wells and the empty well of the 96-well plate at a wavelength of 431 nm was measured. 11) Subtract the absolute fluorescence of the empty well from the absolute fluorescence of each of the first to eighth wells, thereby obtaining the blank removal fluorescence for each of the first to eighth lipid/buffer mixtures; 12) The blank removal fluorescence of each of the first to eighth lipid/buffer mixtures is normalized relative to the blank removal fluorescence of the first lipid/buffer mixture, thereby obtaining a relative fluorescence for each of the first to eighth lipid/buffer mixtures, with the relative fluorescence of the first lipid/buffer mixture set to 1; 13) Plot the relative fluorescence of the first to eighth lipid/buffer mixtures against the pH of the first to eighth lipid/buffer mixtures; 14) Find the pH corresponding to the relative fluorescence of 0.5 from the graph obtained in 13), and determine the pH corresponding to the relative fluorescence of 0.5 as the pKa of the lipid. Determined by, composition.
  3. The composition according to claim 2, wherein the lipid containing the tertiary amine has a pKa in the range of 5.8 to 7.0.
  4. The composition according to claim 1 or 2 , wherein the lipid containing the tertiary amine has a pKa in the range of 6.07 to 7.0.
  5. The composition according to any one of claims 1 to 4 , wherein the liposomes have a diameter in the range of 20 to 220 nm.
  6. The composition according to claim 2, wherein the self-replicating RNA molecule encoding the immunogen has two open reading frames, the first of which encodes alphavirus replicase, and the second of which encodes the immunogen.
  7. The composition according to any one of claims 1 to 6 , wherein the lipid bilayer further comprises polyethylene glycol-conjugated (PEG-conjugated) lipids and/or cholesterol.
  8. The composition according to any one of claims 1 to 7 , wherein the PEG-conjugated lipid is 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)].
  9. The composition according to any one of claims 1 to 8 , wherein the lipid bilayer contains cholesterol in a proportion of 35 mol% to 50 mol% of the total lipids forming the lipid bilayer.
  10. The composition according to any one of claims 1 to 9 , wherein the immunogen comprises two or more immunogens.
  11. The composition according to any one of claims 1 to 10 , wherein the lipid bilayer contains lipids comprising the tertiary amine in a proportion of 40 mol% to 60 mol% of the total lipids forming the lipid bilayer.
  12. The composition according to any one of claims 1 to 11 , wherein the lipid bilayer further comprises 1,2-diastearoyl-sn-glycero-3-phosphocholine.
  13. The composition according to any one of claims 1 to 12 , wherein the lipid bilayer encapsulates 71% to 97.6% of the RNA molecule encoding the immunogen within the aqueous core.
  14. The composition according to any one of claims 1 to 12 , wherein the lipid bilayer encapsulates 85.3% to 97.6% of the RNA molecule encoding the immunogen within the aqueous core.
  15. A pharmaceutical composition comprising the composition described in any one of claims 1 to 14 .
  16. The pharmaceutical composition according to claim 15 for inducing a protective immune response in vertebrates.
  17. A pharmaceutical composition according to claim 15 or 16 for inducing a cell-mediated immune response to the immunogen and/or an antibody response to the immunogen in vivo.
  18. A process for preparing the composition according to claim 1 or 2 , the process comprising, during liposome formation, (i) mixing an RNA molecule encoding the immunogen with a lipid at a pH below the pKa of the lipid but above 4.5; and then (ii) raising the pH above the pKa of the lipid.
  19. A process according to claim 18 , wherein the RNA molecule encoding the immunogen used in step (i) is present in an aqueous solution to be mixed with the organic solution of the lipid to yield a mixture, the mixture is then diluted to form liposomes; and the pH is increased in step (ii) after liposome formation.
  20. Use of the composition according to any one of claims 1 to 14 for producing a pharmaceutical product for inducing a protective immune response in vertebrates.

Description

This application claims the interests of U.S. Provisional Applications No. 61/361,830 (filed July 6, 2010) and No. 61/378,837 (filed August 31, 2010), the entirety of both aforementioned U.S. Provisional Applications being incorporated herein by reference for all purposes. (Technical field) This invention relates to the field of nonviral delivery of RNA for immune purposes. (Background technology) The delivery of nucleic acids to immunize animals has been a goal for several years. Various approaches have been tested, including the use of DNA or RNA, the use of viral or nonviral delivery vehicles (or even without a delivery vehicle in "naked" vaccines), the use of replicated or non-replicated vectors, or the use of viral or nonviral vectors. There is a need for further improved nucleic acid vaccines. Figure 1 shows a gel containing stained RNA. The lanes represent (1) marker, (2) bare replicon, (3) replicon after RNase treatment, (4) replicon encapsulated in liposomes, (5) liposomes after RNase treatment, and (6) liposomes treated with RNase and then subjected to phenol/chloroform extraction.Figure 2 is an electron microscope image of a liposome.Figure 3 shows the structures of DLinDMA, DLenDMA, and DODMA.Figure 4 shows a gel containing stained RNA. The lanes represent (1) a marker, (2) bare replicons, (3) replicons encapsulated in liposomes, and (4) liposomes treated with RNase and then subjected to phenol/chloroform extraction.Figure 5 shows protein expression on days 1, 3, and 6 after RNA delivery as vilion-packaged replicons (squares), as naked RNA (diamonds), or in liposomes (+ = 0.1 μg, × = 1 μg).Figure 6 shows protein expression on days 1, 3, and 6 after delivery of four different doses of liposome-encapsulated RNA.Figure 7 shows the anti-IgG titers in animals given virion-packaged replicons (VRP or VSRP), 1 μg of bare RNA, and 1 μg of liposome-encapsulated RNA.Figure 8 shows the anti-IgG titers in animals given VRP, 1 μg of bare RNA, and 0.1 g or 1 μg of liposome-encapsulated RNA.Figure 9 shows the neutralizing antibody titers in animals given either VRP or 0.1 g or 1 μg of liposome-encapsulated RNA.Figure 10 shows the expression levels after delivery of the replicon as bare RNA (circles), liposome-encapsulated RNA (triangles and squares), or lipoplex (inverted triangle).Figure 11 shows the F-specific IgG titer (2 weeks after the second dose) after delivery of the replicon as naked RNA (0.01–1 μg), as liposome-encapsulated RNA (0.01–10 μg), or as packaged virions (VRP, 10⁶ infection units, or IU).Figure 12 shows the F-specific IgG titers (circles) and PRNT titers (squares) after delivery of the replicon as naked RNA (1 μg), as liposome-encapsulated RNA (0.1 or 1 μg), or as packaged as a virion (VRP, 10⁶ IU). Titers in naive mice are also shown. The solid line represents the geometric mean.Figure 13 shows intracellular cytokine production four weeks after the second dose, following restimulation with a synthetic peptide representing the major epitope in the F protein. The y-axis represents the % cytokine+ of CD8+ and CD4-.Figure 14 shows the F-specific IgG titers (mean log 10 titer ± standard deviation) of calves 63 days (Figure 14A) and 210 days (Figure 14B) after immunization. The three lines are easily distinguishable at day 63, from bottom to top: PBS-negative control; liposome-delivered RNA; and "Triangle 4" product.Figure 14 shows the F-specific IgG titers (mean log 10 titer ± standard deviation) of calves 63 days (Figure 14A) and 210 days (Figure 14B) after immunization. The three lines are easily distinguishable at day 63, from bottom to top: PBS-negative control; liposome-delivered RNA; and "Triangle 4" product.Figure 15 shows the SEAP expression (relative intensity) on day 6 against the pKa of the lipids used in the liposomes described above. Circles indicate the levels for liposomes with DSPCs, and squares indicate the levels for liposomes without DSPCs; occasionally, the squares and circles overlap, so that only the squares are visible for a given pKa.Figure 16 shows the expression of anti-F titer (RV01, 100%) two weeks after the first administration of the replicon encoding the F protein. The titers are plotted against pKa in the same manner as in Figure 15. Stars indicate RV02 using cationic lipids with higher pKa than other lipids. Triangles indicate data for liposomes lacking DSPC; circles indicate data for liposomes containing DSPC.Figure 17 shows, from left to right, the total IgG titers after replicon delivery in liposomes using RV01, RV16, RV17, RV18, or RV19. The bars represent the mean values. In each case, the upper bar is 2wp2 (i.e., 2 weeks after the second dose), while the lower bar is 2wp1.Figure 18 shows the IgG titers in 13 mouse groups. Each circle represents an individual mouse, and the solid line shows the geometric mean. The horizontal dashed line is the detection limit of the assay. The 13 groups are labeled A through M from left to right, as shown below.Figure 19 sho