CA-3301230-A1 - BUFFERS FOR STABILZATION OF LENTIVIRAL PREPARATIONS

Abstract

The invention provides lentiviral preparations containing a sulfonic acid buffer, such as 1,4- piperazinediethanesulfonic acid (PIPES), 2-(N-morpholino)ethanesulfonic acid (MES), and 3- morpholinopropane-1 -sulfonic acid (MOPS), a sodium citrate buffer, or a phosphate buffer. The invention additionally encompasses methods of lentiviral purification as well as methods of transducing human cells.

Inventors

• Amitabha DEB
• Eugene NEBELITSKY
• Vladimir Slepushkin

Assignees

• NOVARTIS AG
• THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA

Dates

Publication Date

20260302

Application Date

20161118

Priority Date

20151119

Claims (1)

1. <pat:ClaimStatement>CLAIMS</pat:ClaimStatement> <pat:Claims com:id="claims"> <pat:Claim com:id="CLM-00001"> <pat:ClaimNumber>1</pat:ClaimNumber> <pat:ClaimText>1. An aqueous composition comprising a lentiviral vector, a 1,4-piperazinediethanesulfonic acid (PIPES) buffer, and a salt. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00002"> <pat:ClaimNumber>2</pat:ClaimNumber> <pat:ClaimText>2. The aqueous composition of claim 1, wherein said PIPES buffer is present at a concentration of from about 10 mM to about 50 mM. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00003"> <pat:ClaimNumber>3</pat:ClaimNumber> <pat:ClaimText>3. The aqueous composition of claim 2, wherein said PIPES buffer is present at a concentration of about 20 mM. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00004"> <pat:ClaimNumber>4</pat:ClaimNumber> <pat:ClaimText>4. The aqueous composition of any one of claims 1-3, wherein the pH of said aqueous composition is from about 6.0 to about 7.0. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00005"> <pat:ClaimNumber>5</pat:ClaimNumber> <pat:ClaimText>5. The aqueous composition of claim 4, wherein the pH of said aqueous composition is about </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00006"> <pat:ClaimNumber>6</pat:ClaimNumber> <pat:ClaimText>6. The aqueous composition of any one of claims 1-5, wherein said salt is selected from the group consisting of sodium chloride, magnesium chloride, and calcium chloride. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00007"> <pat:ClaimNumber>7</pat:ClaimNumber> <pat:ClaimText>7. The aqueous composition of claim 6, wherein said salt is sodium chloride. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00008"> <pat:ClaimNumber>8</pat:ClaimNumber> <pat:ClaimText>8. The aqueous composition of any one of claims 1-7, wherein the concentration of said salt in said aqueous composition is from about 25 mM to about 150 mM. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00009"> <pat:ClaimNumber>9</pat:ClaimNumber> <pat:ClaimText>9. The aqueous composition of claim 8, wherein the concentration of said salt in said aqueous composition is about 50 mM. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00010"> <pat:ClaimNumber>10</pat:ClaimNumber> <pat:ClaimText>10. The aqueous composition of claim 8, wherein the concentration of said salt in said aqueous composition is about 75 mM. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00011"> <pat:ClaimNumber>11</pat:ClaimNumber> <pat:ClaimText>11. The aqueous composition of any one of claims 1-10, wherein said aqueous composition comprises 20 mM PIPES and 75 mM sodium chloride, and wherein said aqueous composition has a pH of about 6.5. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00012"> <pat:ClaimNumber>12</pat:ClaimNumber> <pat:ClaimText>12. The aqueous composition of any one of claims 1-11, wherein said aqueous composition further comprises a carbohydrate. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00013"> <pat:ClaimNumber>13</pat:ClaimNumber> <pat:ClaimText>13. The aqueous composition of claim 12, wherein said carbohydrate is a non-reducing carbohydrate. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00014"> <pat:ClaimNumber>14</pat:ClaimNumber> <pat:ClaimText>14. The aqueous composition of claim 13, wherein said non-reducing carbohydrate is selected from the group consisting of sucrose and trehalose. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00015"> <pat:ClaimNumber>15</pat:ClaimNumber> <pat:ClaimText>15. The aqueous composition of any one of claims 12-14, wherein said carbohydrate is present at a concentration of from about 1% to about 10% by weight per volume of said aqueous composition. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00016"> <pat:ClaimNumber>16</pat:ClaimNumber> <pat:ClaimText>16. The aqueous composition of claim 15, wherein said carbohydrate is present at a concentration of from about 2% to about 5% by weight per volume of said aqueous composition. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00017"> <pat:ClaimNumber>17</pat:ClaimNumber> <pat:ClaimText>17. The aqueous composition of claim 16, wherein said carbohydrate is present at a concentration of about 2.5% by weight per volume of said aqueous composition. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00018"> <pat:ClaimNumber>18</pat:ClaimNumber> <pat:ClaimText>18. The aqueous composition of any one of claims 12-17, wherein said aqueous composition comprises 20 mM PIPES, 75 mM sodium chloride, and 2.5% sucrose by weight per volume of said aqueous composition, and wherein said aqueous composition has a pH of about 6.5. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00019"> <pat:ClaimNumber>19</pat:ClaimNumber> <pat:ClaimText>19. The aqueous composition of any one of claims 1-18, wherein the osmolality of said aqueous composition is from about 270 mOsm/kg to about 330 mOsm/kg. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00020"> <pat:ClaimNumber>20</pat:ClaimNumber> <pat:ClaimText>20. The aqueous composition of claim 19, wherein the osmolality of said aqueous composition is from about 275 mOsm/kg to about 300 mOsm/kg. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00021"> <pat:ClaimNumber>21</pat:ClaimNumber> <pat:ClaimText>21. The aqueous composition of claim 20, wherein the osmolality of said aqueous composition is about 285 mOsm/kg. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00022"> <pat:ClaimNumber>22</pat:ClaimNumber> <pat:ClaimText>22. The aqueous composition of any one of claims 1-21, wherein said lentiviral vector is present at a concentration of from about 2 x 10⁸ transducing units per milliliter (TU/mL) to about 1 x 10⁹ TU/mL. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00023"> <pat:ClaimNumber>23</pat:ClaimNumber> <pat:ClaimText>23. The aqueous composition of claim 22, wherein said lentiviral vector is present at a concentration of from about 3 x 10⁸ TU/mL to about 5 x 10⁸ TU/mL. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00024"> <pat:ClaimNumber>24</pat:ClaimNumber> <pat:ClaimText>24. The aqueous composition of any one of claims 1-23, wherein said lentiviral vector is a recombinant human immunodeficiency virus. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00025"> <pat:ClaimNumber>25</pat:ClaimNumber> <pat:ClaimText>25. The aqueous composition of any one of claims 1-24, wherein said lentiviral vector comprises vesicular stomatitis virus G (VSV-G) protein. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00026"> <pat:ClaimNumber>26</pat:ClaimNumber> <pat:ClaimText>26. The aqueous composition of claim 25, wherein said VSV-G protein is present on the surface of said lentiviral vector. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00027"> <pat:ClaimNumber>27</pat:ClaimNumber> <pat:ClaimText>27. The aqueous composition of any one of claims 1-26, wherein said lentiviral vector comprises a transgene. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00028"> <pat:ClaimNumber>28</pat:ClaimNumber> <pat:ClaimText>28. The aqueous composition of claim 27, wherein said transgene encodes a protein. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00029"> <pat:ClaimNumber>29</pat:ClaimNumber> <pat:ClaimText>29. The aqueous composition of claim 28, wherein said protein comprises a chimeric antigen receptor (CAR). </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00030"> <pat:ClaimNumber>30</pat:ClaimNumber> <pat:ClaimText>30. The aqueous composition of claim 29, wherein said CAR comprises, in an N-terminal to C- terminal direction, an antigen binding domain, a transmembrane domain, and one or more signaling domains. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00031"> <pat:ClaimNumber>31</pat:ClaimNumber> <pat:ClaimText>31. The aqueous composition of claim 30, wherein said signaling domain comprises one or more primary signaling domains. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00032"> <pat:ClaimNumber>32</pat:ClaimNumber> <pat:ClaimText>32. The aqueous composition of claim 30 or 31, wherein said signaling domains comprise one or more costimulatory signaling domains. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00033"> <pat:ClaimNumber>33</pat:ClaimNumber> <pat:ClaimText>33. The aqueous composition of claim 31, wherein one of said one or more primary signaling domains comprises a CD3-zeta stimulatory domain. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00034"> <pat:ClaimNumber>34</pat:ClaimNumber> <pat:ClaimText>34. The aqueous composition of claim 32 or 33, wherein one or more of said costimulatory signaling domains comprises an intracellular domain selected from a costimulatory protein selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CDS, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, and a ligand that specifically binds with CD83. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00035"> <pat:ClaimNumber>35</pat:ClaimNumber> <pat:ClaimText>35. The aqueous composition of claim 34, wherein said one or more of said costimulatory signaling domains comprises the 4-1BB (CD137) costimulatory domain. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00036"> <pat:ClaimNumber>36</pat:ClaimNumber> <pat:ClaimText>36. The aqueous composition of claim 34 or 35, wherein said one or more of said costimulatory domains comprises the CD28 costimulatory domain. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00037"> <pat:ClaimNumber>37</pat:ClaimNumber> <pat:ClaimText>37. The aqueous composition of any one of claims 30-36, wherein said antigen binding domain is an scFv. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00038"> <pat:ClaimNumber>38</pat:ClaimNumber> <pat:ClaimText>38. The aqueous composition of any one of claims 30-37, wherein said antigen binding domain binds to an antigen selected from the group consisting of CD19; CD123; CD22; CD30; CD171; CS-1; C- type lectin-like molecule-1, CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3; TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAcα-Ser/Thr)); prostate-specific membrane antigen (PSMA); Receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-Like Tyrosine Kinase 3 (FLT3); Tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); Interleukin-13 receptor subunit alpha-2; mesothelin; Interleukin 11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); Protease Serine 21; vascular endothelial growth factor receptor 2 (VEGFR2); Lewis(Y) antigen; CD24; Platelet-derived growth factor receptor beta (PDGFR- beta); Stage-specific embryonic antigen-4 (SSEA-4); CD20; Folate receptor alpha; Receptor tyrosine- protein kinase ERBB2 (Her2/neu); Mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); Prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); Ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr- abl); tyrosinase; ephrin type-A receptor 2 (EphA2); Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3; transglutaminase 5 (TGS5); high molecular weight-melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); Folate receptor beta; tumor endothelial marker 1 (TEM1/CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); Polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion of globoH glycoceramide (GloboH); mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K 9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumor protein (WT1); Cancer/testis antigen 1 (NY-ESO-1); Cancer/testis antigen 2 (LAGE-1a); Melanoma-associated antigen 1 (MAGE-A1); ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X Antigen Family, Member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; surviving; telomerase; prostate carcinoma tumor antigen-1, melanoma antigen recognized by T cells 1; Rat sarcoma (Ras) mutant; human Telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML- IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetyl glucosaminyl- transferase V (NA17); paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin B1; v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family Member C (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 1B1 (CYP1B1); CCCTC- Binding Factor (Zinc Finger Protein)-Like, Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3); Paired box protein Pax-5 (PAX5); proacrosin binding protein sp32 (OY-TES1); lymphocyte- specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); Receptor for Advanced Glycation Endproducts (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papilloma virus E6 (HPV E6); human papilloma virus E7 (HPV E7); intestinal carboxyl esterase; heat shock protein 70-2 mutated (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1). </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00039"> <pat:ClaimNumber>39</pat:ClaimNumber> <pat:ClaimText>39. The aqueous composition of claim 38, wherein said antigen binding domain binds to CD19, mesothelin, or CD123. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00040"> <pat:ClaimNumber>40</pat:ClaimNumber> <pat:ClaimText>40. The aqueous composition of any one of claims 29-39, wherein said CAR comprises an anti- CD19 antibody or a fragment thereof, a 4-1BB (CD137) transmembrane domain, and a CD3-zeta signaling domain. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00041"> <pat:ClaimNumber>41</pat:ClaimNumber> <pat:ClaimText>41. The aqueous composition of any one of claims 1-40, wherein said aqueous composition is free of one or more proteins selected from the group consisting of human serum albumin (HSA), recombinant human serum albumin (rHSA), bovine serum albumin (BSA), and a lipoprotein. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00042"> <pat:ClaimNumber>42</pat:ClaimNumber> <pat:ClaimText>42. The aqueous composition of claim 41, wherein said aqueous composition is free of HSA, rHSA, BSA, and lipoproteins. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00043"> <pat:ClaimNumber>43</pat:ClaimNumber> <pat:ClaimText>43. The aqueous composition of any one of claims 1-42, wherein said lentiviral vector is produced in cells cultured in the absence of serum. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00044"> <pat:ClaimNumber>44</pat:ClaimNumber> <pat:ClaimText>44. The aqueous composition of any one of claims 1-43, wherein said lentiviral vector is characterized by a hydrodynamic radius of <math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mrow><mn>100</mn><mo>±</mo><mn>25</mn></mrow><annotation encoding="application/x-tex">100 \pm 25</annotation></semantics></math> nm as measured by dynamic light scattering (DLS). </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00045"> <pat:ClaimNumber>45</pat:ClaimNumber> <pat:ClaimText>45. The aqueous composition of claim 44, wherein said lentiviral vector maintains said hydrodynamic radius of 100 <math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mo>±</mo><annotation encoding="application/x-tex">\pm</annotation></semantics></math> 25 nm within a temperature range of from 25 <math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><msup><mi></mi><mo>∘</mo></msup><annotation encoding="application/x-tex">^{\circ}</annotation></semantics></math>C to 55 <math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><msup><mi></mi><mo>∘</mo></msup><annotation encoding="application/x-tex">^{\circ}</annotation></semantics></math>C. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00046"> <pat:ClaimNumber>46</pat:ClaimNumber> <pat:ClaimText>46. The aqueous composition of any one of claims 1-45, wherein said lentiviral vector is characterized by a polydispersity of from 10% to 25%. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00047"> <pat:ClaimNumber>47</pat:ClaimNumber> <pat:ClaimText>47. The aqueous composition of claim 46, wherein said lentiviral vector maintains said polydispersity of from 10% to 25% within a temperature range of from 25 <math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><msup><mi></mi><mo>∘</mo></msup><annotation encoding="application/x-tex">^{\circ}</annotation></semantics></math>C to 55 <math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><msup><mi></mi><mo>∘</mo></msup><annotation encoding="application/x-tex">^{\circ}</annotation></semantics></math>C. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00048"> <pat:ClaimNumber>48</pat:ClaimNumber> <pat:ClaimText>48. The aqueous composition of any one of claims 1-47, wherein said lentiviral vector maintains a concentration after 3 freeze/thaw cycles of from about 70% to about 100% relative to the concentration of said lentiviral vector in said aqueous composition prior to said freeze/thaw cycles, wherein each of said freeze/thaw cycles comprises freezing said aqueous composition and subsequently allowing said aqueous composition to thaw at room temperature. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00049"> <pat:ClaimNumber>49</pat:ClaimNumber> <pat:ClaimText>49. The aqueous composition of claim 48, wherein said lentiviral vector maintains said concentration of from about 70% to about 100% after 6 of said freeze/thaw cycles. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00050"> <pat:ClaimNumber>50</pat:ClaimNumber> <pat:ClaimText>50. The aqueous composition of claim 49, wherein said lentiviral vector maintains said concentration of from about 70% to about 100% after 9 of said freeze/thaw cycles. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00051"> <pat:ClaimNumber>51</pat:ClaimNumber> <pat:ClaimText>51. An aqueous composition comprising a lentiviral vector, a buffer selected from the group consisting of a phosphate buffer, a sodium citrate buffer, a 2-(N-morpholino)ethanesulfonic acid (MES) buffer, a 3-morpholinopropane-1-sulfonic acid (MOPS) buffer, and a salt. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00052"> <pat:ClaimNumber>52</pat:ClaimNumber> <pat:ClaimText>52. The aqueous composition of claim 51, wherein said salt is selected from the group consisting of sodium chloride, magnesium chloride, and calcium chloride. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00053"> <pat:ClaimNumber>53</pat:ClaimNumber> <pat:ClaimText>53. The aqueous composition of claim 51 or 52, wherein said aqueous composition further comprises a non-reducing carbohydrate selected from the group consisting of sucrose and trehalose. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00054"> <pat:ClaimNumber>54</pat:ClaimNumber> <pat:ClaimText>54. A method of purifying a lentiviral vector, said method comprising passing the aqueous composition of any one of claims 1-53 through a filter, thereby producing an aqueous composition that is substantially free of microorganisms. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00055"> <pat:ClaimNumber>55</pat:ClaimNumber> <pat:ClaimText>55. The method of claim 54, wherein said filter comprises a plurality of pores, and wherein said pores have a diameter of about 0.2 µm. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00056"> <pat:ClaimNumber>56</pat:ClaimNumber> <pat:ClaimText>56. The method of claim 55, wherein said aqueous composition that is substantially free of microorganisms comprises said lentiviral vector at a concentration of about 80% relative to the concentration of said lentiviral vector in said aqueous composition prior to said contacting. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00057"> <pat:ClaimNumber>57</pat:ClaimNumber> <pat:ClaimText>57. A method of purifying a lentiviral vector, said method comprising: a. contacting the aqueous composition of any one of claims 1-53 with a material comprising a plurality of particles; and b. separating substances that flow through said material from substances that remain within said material, thereby producing an aqueous composition that is enriched with said lentiviral vector. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00058"> <pat:ClaimNumber>58</pat:ClaimNumber> <pat:ClaimText>58. A method of purifying a lentiviral vector, said method comprising contacting the aqueous composition of any one of claims 1-53 with a nuclease, thereby producing an aqueous composition that is substantially free of contaminating polynucleotides. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00059"> <pat:ClaimNumber>59</pat:ClaimNumber> <pat:ClaimText>59. A method of expressing a transgene in a cell, said method comprising contacting said cell with the aqueous composition of any one of claims 1-53. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00060"> <pat:ClaimNumber>60</pat:ClaimNumber> <pat:ClaimText>60. The method of claim 59, wherein said cell is a mammalian cell. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00061"> <pat:ClaimNumber>61</pat:ClaimNumber> <pat:ClaimText>61. The method of claim 60, wherein said mammalian cell is a T cell. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00062"> <pat:ClaimNumber>62</pat:ClaimNumber> <pat:ClaimText>62. The method of claim 61, wherein said T cell is a human T cell. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00063"> <pat:ClaimNumber>63</pat:ClaimNumber> <pat:ClaimText>63. The method of claim 62, wherein said human T cell is a 293T cell, a Jurkat T cell, or a primary human T cell. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00064"> <pat:ClaimNumber>64</pat:ClaimNumber> <pat:ClaimText>64. A kit comprising the aqueous composition of any one of claims 1-53 and a package insert. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00065"> <pat:ClaimNumber>65</pat:ClaimNumber> <pat:ClaimText>65. The kit of claim 64, wherein said package insert instructs a user of said kit to express a transgene in a cell according to the method of any one of claims 59-63. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00066"> <pat:ClaimNumber>66</pat:ClaimNumber> <pat:ClaimText>66. The kit of claim 65, wherein said kit further comprises a reagent that can be used to culture the cell of claim 65. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00067"> <pat:ClaimNumber>67</pat:ClaimNumber> <pat:ClaimText>67. Use of an aqueous composition of any one of claims 1-53 in a method for delivering a viral vector, which optionally comprises a transgene, into a cell of a subject, the method comprising administering the composition to the subject. </pat:ClaimText> </pat:Claim> </pat:Claims>

Description

BUFFERS FOR STABILIZATION OF LENTIVIRAL PREPARATIONS Cross-Reference to Related Applications This application is a division of CA 3,005,125 filed November 18, 2016, and claims priority to US 62/257,444 filed November 19, 2015. Field of the Invention The invention relates to lentiviral preparations that exhibit improved transducing capabilities and storage stability, as well as to methods of heterologous gene expression in target cells. Background of the Invention With advances in gene therapy technologies, the use of therapeutic viral vectors represents an increasingly effective paradigm for treating human diseases. Among the viral vectors available for gene therapy applications are lentiviral vectors. Such vectors include reconstructed viral vector systems derived from human immunodeficiency virus-1 (HIV-1) and are capable of introducing a gene of interest into animal and human primary cells or cell lines. The genomes of lentiviral vectors include a coding strand of RNA, which is reverse-transcribed into DNA upon entering the cytoplasm of a host cell by a viral reverse transcriptase so as to form a DNA pre-integration complex. This complex is transported into the nucleus of the host cell, where a portion of the viral DNA is subsequently integrated into the host cell genome. The integrated DNA can then be transcribed into RNA, such as protein-coding mRNA, which can ultimately be exported to the cytoplasm for subsequent expression of a protein of interest. Lentiviral vector-mediated gene expression can be used to achieve continuous and stable protein production, because the gene of interest has been integrated into a host cell's genome and is thus replicated upon division of the cell. Lentiviral vectors can effectively infect non-dividing cells as well as those actively progressing through the cell cycle. In contrast, other viral vectors, such as adenoviral vectors, adeno-associated viral vectors, and classical retroviral vectors, are only capable of infecting dividing cells. Tissues and cells in which lentiviral vector-mediated chronic expression of a gene of interest can occur include the brain, liver, muscle cells, retina, hematopoietic stem cells, marrow mesenchymal stem cells, and macrophages, among others. The production of lentiviral vectors has been hindered by several challenges, one of which is low stability of the vectors. The manufacturing operation of lentiviral vectors includes several steps: production, purification, storage, and application of gene transfer (Carmo et al., J. Gene Med. 11:670-678, 2009). Lentiviral vectors are susceptible to inactivation during these processes, which can contribute to diminished final quality and efficacy of the vector preparation. In previous studies, it has been shown that one mechanism by which viral vectors are inactivated is by the loss of viral capacity to perform reverse transcription (Carmo et al., Hum. Gene Ther. 20:1168-76, 2009; and Carmo et al., J. Gene Med. 10:383- 391, 2008). Moreover, there remains a need for methods to stabilize lentiviral vector preparations so as to prevent irreversible aggregation that can be accompanied by loss of infectivity. Additionally, during the purification of lentiviral vectors, stabilizing components are removed from the lentiviral preparation, which can cause the vector to become increasingly unstable. Therefore, there is also a need for lentiviral formulations that preserve vector stability throughout the purification process. During purification and storage, vectors are often stored at 4°C (Rodrigues et al., J. Biotechnol. 127:520-541, 2007). It has been reported that lentiviral vectors have an additional need for stabilizing components, such as human serum albumin (HSA) (Carmo et al., J. Gene Med. 11:670-678, 2009). This is in sharp contrast to gamma-retroviruses, where simply adding exogenous proteins brings back the stability comparable to cell culture supernatant. Lipoproteins are complex structures composed of several lipids, including cholesterol, phospholipids, and proteins (Olson, J. Nutr. 128:S439-S443, 1998). They act as lipid transporters in blood along with HSA. It is possible that a lipoprotein-HSA structure forms a protective arrangement around the membrane of lentiviral vectors (Carmo et al., J. Gene Med. 11:670- 678, 2009). Because albumin is also known to associate tightly with cell surfaces (Dziarski et al., J. Biol. Chem. 269:20431-20436, 1994), these lipoprotein/HSA complexes can associate with the membrane of the vector, which is similar to a cell membrane. This association may provide protection to their structure and prevent conformational changes more efficiently than HSA alone. In order to ensure stability during storage, stocks of infective viral vectors have commonly been stored at low temperatures (e.g., at -80 °C) due to their complexity. It has been suggested that lipid- enveloped viruses survive well at temperatures below -60 ℃, and that storage at -20 ℃ or 4 ℃ should only be used