ASSEMBLY ACTIVATING PROTEIN (AAP) AND ITS USE FOR THE MANUFACTURE OF PARVOVIRUS PARTICLES ESSENTIALLY CONSISTING OF VP3
20170226160 · 2017-08-10
Inventors
- Florian Sonntag (Heidelberg, DE)
- Juergen Kleinschmidt (Bammental, DE)
- Markus Hoerer (Planegg, DE)
- Kerstin LUX (Muenchen, DE)
Cpc classification
A61P1/04
HUMAN NECESSITIES
C12N7/00
CHEMISTRY; METALLURGY
A61P29/00
HUMAN NECESSITIES
A61P31/00
HUMAN NECESSITIES
C12N2750/14143
CHEMISTRY; METALLURGY
C12N2750/14122
CHEMISTRY; METALLURGY
A61P9/10
HUMAN NECESSITIES
A61P1/00
HUMAN NECESSITIES
A61P25/28
HUMAN NECESSITIES
A61P37/06
HUMAN NECESSITIES
International classification
C12N7/00
CHEMISTRY; METALLURGY
Abstract
The present invention relates to nucleic acids encoding the novel parvoviral protein “assembly activating protein” S(AAP), the encoded polypeptides, methods of producing the polypeptides, antibodies specific for AAP, the use of the nucleic acids for the preparation of the polypeptides, the use of the nucleic acids or the polypeptides for the preparation of the parvoviral particle and methods of producing parvoviral particles essentially consisting of VP3 by providing in addition to the coding sequence of the parvoviral structural protein VP3 a sequence fragment Z/a nucleic acid encoding AAP in the cell and expressing VP3 and fragment Z under control of a rep-independent promoter. Furthermore, the present invention relates to parvoviral particles essentially consisting of VP3 and/or obtainable by the above method as well as expression cassettes comprising (i) a heterologous promoter and (ii) VP3 coding sequence and/or fragment Z. The present invention further relates to a medicament, particularly a vaccine, comprising the parvoviral particles or expression cassettes and their use.
Claims
1-47. (canceled)
48. A parvoviral particle consisting essentially of VP3, i. wherein the VP3 optionally comprises one or more mutation(s), and ii. wherein the VP3 does not contain a heterologous nuclear localization signal, and iii. wherein the particle does not contain any of the functional Rep proteins, particularly Rep40, Rep52, Rep68 and Rep78.
49. The parvoviral particle according to claim 48, wherein the capsid consists only of VP3.
50. The parvoviral particle according to claim 48, wherein the mutation(s) of VP3 is/are: a) one or more mutation(s) selected from the group consisting of one or more deletion(s), one or more insertion(s), one or more substitution(s), and a combination thereof; b) one or more silent mutation(s); c) one or more mutations located on the surface of a VP3 virus-like particle (VLP); d) one or more mutation(s) located at the N-terminus of VP3; e) one or more insertions at one or more positions selected from the group consisting of I-261, I-266, I-381, I-447, I-448, I-453, I-459, I-471, I-534, I-570, I-573, I-584, I-587, I-588, I-591, I-657, I-664, I-713, and I-716; or wherein two insertions are made at two positions selected from the group consisting of I-261, I-453, I-534, I-570, I-573, and I-587, wherein mutations are made into position 453 in combination with an insertion in position 587 and in combination with an additional mutation; f) at least one epitope heterologous to the virus, particularly wherein the epitope of the VP3 protein is a B-cell epitope and/or particularly wherein the B-cell epitope is inserted into I-453 and/or I-587, especially into I-453 and/or I-587 of AAV1, AAV2 or AAV4; q) wherein the VP3 is included in a fusion protein; h) at least one tag useful for binding to a ligand; and/or i) at least one further mutation.
51-54. (canceled)
55. A pharmaceutical composition comprising the parvoviral particle of claim 48.
56. The pharmaceutical composition of claim 55, further comprising one or more excipients.
57. The pharmaceutical composition of claim 55, wherein the pharmaceutical composition is a vaccine.
58. The pharmaceutical composition of claim 57, wherein the vaccine further comprises one or more adjuvants.
59. A method for preventing or treating an autoimmune disease, an infectious disease, a tumor disease, an allergic disease, a metabolic disease, a (chronic) inflammatory disease, a neurological disease, addiction, or an ophthalmological disease, the method comprising administering the pharmaceutical composition of claim 55 to a patient in need thereof.
60. The method of claim 59, wherein the autoimmune disease and/or a chronic inflammatory disease is selected from the group consisting of rheumatoid arthritis, psoriasis and Crohn's disease.
61. The method of claim 59, wherein the tumor disease is eligible for treatment with a monoclonal antibody.
62. The method of claim 59, wherein the allergic disease is selected from the group consisting of asthma, allergy and allergic rhinitis.
63. The method of claim 59, wherein the neurological disease is Alzheimer's disease.
64. The method of claim 59, wherein the metabolic disease is atherosclerosis
65. The method of claim 59, wherein the ophthalmological disease is age-related macular degeneration.
66-114. (canceled)
Description
FIGURES
[0197]
[0198] The coding DNA for the cap gene is shown in the first line, the Cap proteins VP1, VP2 and VP3 in the following ones. Nucleotide numbers correspond to the genome sequence of AAV-2 given by Ruffing et al. (1994) accessible from NCBI (number of entree: NC_001401). Numbering of amino acid (AA) sequences according to VP1 of AAV2 (Girod et al. 1999). EcoNI and BsiWI restriction sites are marked. Not to scale.
[0199]
[0200] The nucleotide sequences of fragment Z of the parvoviruses AAV1 (NC_002077), AAV2 (AF043303), AAV3b (AF028705), AAV4 (U89790), AAV5 (NC_006152), AAV6 (AF028704), AAV7 (AF513851), AAV8 (AF513852), AAV10 (AY631965), AAV11 (AY631966), and b-AAV (NC_005889) are given (numbers of nucleotide entrees according to NCBI are given in brackets). +1 indicates the position of the first nucleotide coding for the ATG start codon of VP3. The 44 nucleotides upstream and 242 nucleotides downstream of the +1 position are shown. The ATG start codon of VP3 is underlined.
[0201]
[0202] Six possible expression constructs differing in the set-up of the fragment Z sequence and VP3 cds are shown by different boxes as indicated. In the cis situation they are expressed under the same one promoter whereas in trans two separate promoters drive their expression, as indicated by the circle. +1 indicates the position of the first nucleotide coding for the ATG start codon of VP3. The DNA of fragment Z comprising at least 44 nucleotides upstream and more than 242 nucleotides downstream of the +1 position are boxed (compare
[0203]
[0204] Schematic representation of the rep and cap genes in the parvovirus genome. The position of the restriction sites R1 to R5 used for cloning of the different expression constructs, as well as the positions of the translation start codons of the three capsid proteins are marked. Not to scale
[0205]
[0206]
[0207]
[0208]
[0209]
[0210] Western blot analysis of purified wt AAV and capsids derived from pVP3/2696 or pVP3/2809 trans-complemented with pVP2N-gfp. Detection of VP1 and VP2 occurred with antibody A69. Different amounts of capsids as indicated were loaded to the gel for a qualitative estimation of the ratio of different signals (VP2tru=truncated VP2).
[0211]
[0212]
[0213]
[0214]
[0215]
[0216]
[0217] Localization of capsid proteins expressed from different constructs in HeLa cells was visualized by double immunofluorescence using a polyclonal rabbit antiserum detecting total capsid proteins (VPs) and monoclonal antibody A20 detecting assembled capsids. The transfected plasmids are indicated at the left margin.
[0218] Immunofluorescence staining of transfected HeLa cells with the A20 antibody showed that the VP protein of mutant pCMV-VP3/2696RKR168-170AAA was as efficient in capsid assembly as wt AAV. For the construct pCMV-VP3/2696RKR168-170AAA the postulated NLS was mutated by converting the RKR peptide (AA 168-170).
[0219]
[0220]
[0221]
[0222]
[0223]
[0224]
[0225] Western blot analysis of expressed VP proteins in crude lysates of 293 cells transfected with different AAV1 constructs: pCI_VP2/2539_AAV1, pCI_VP3/2539_AAV1 mutACG, pCI_VP3/2634_AAV1 mutACG and pUCAV1. Detection of VP proteins was performed using the B1 antibody (dilution: 1:250) (Progen Heidelberg, Germany) and subsequent the secondary antibody anti mouse IgG-HRP 1:2500 (Dianova, Hamburg, Germany).
[0226] 2E10 particles per construct were loaded according to AAV1 titration by an AAV1 capsid ELISA (Progen Heidelberg, Germany).
[0227] The Western Blot shows that construct pUCAV1 expresses the three capsid proteins VP1, VP2 and VP3 (lane 5) whereas pCI_VP2/2539_AAV1 leads to expression of VP2 and VP3 (lane 2) and within lysates of cells transfected with pCI_VP3/2539_AAV1 mutACG and pCI_VP3/2634_AAV1 mutACG only VP3 could be detected (lane 3 and 4).
[0228]
[0229] Western blot analysis of cell extracts transfected with VP3 expression construct of AAV2 pCMV-VP3/2809 or of AAV1 pCMV-AAV1VP3/2828 (indicated in the figure as AAV2 or AAV1, respectively) with or without cotransfection of pVP2N-gfp. AAV1 and AAV2 VP3 was detected by the antibody B1 (Progen, Heidelberg, Germany) which recognizes an epitope completely conserved between AAV1 and AAV2. The VP2N-gfp protein was detected by antibody A69 (Progen, Heidelberg, Germany).
[0230]
[0231]
[0232] For cloning of the constructs pCI-VP2mutACG, pCMV-NLS-VP3, and pCMV-VP3/2696 please refer to elsewhere.
[0233]
[0234] Subsequently, 293-T cells were lysed in the medium by three rounds of freeze (−80° C.) and thaw (37° C.) cycles. The lysate (3 ml total volume) was cleared by centrifugation and the VLP capsid titer was determined using a commercially available ELISA (AAV Titration ELISA, Progen). Average values of 4 to 6 independent transfections per construct are indicated with respective error bars.
[0235] Notably, particle production efficacy with construct pCMC-NLS-VP3 was below the detection limit (about 1E+09/ml) and, therefore, at least 3-4 logs lower compared to the best VP3 particle production vectors described in this invention (pCI-VP2mutACG and pCMV-VP3/2696).
[0236]
[0237] Subsequently, supernatant of 293-T cells was removed, cells were rinsed with PBS and finally lysed in 300 μl RIPA buffer (25 mM Tris.Cl pH 7.4, 150 mM NaCl, 1% IGEPAL, 1% Na.DOC, 0.1% SDS). 100 μl 3×Geba sample buffer (Gene Bio-Application Ltd) and 25 mM DTT were added, and samples were heated at 95° C. for 10 min. Samples were centrifuged and 30 μl cleared supernatant were subjected to SDS page (10% GeBa gels, Gene Bio-Application Ltd). Proteins were transferred to a nitrocellulose membrane (1 h, 230 mA) which was blocked for 1 h at RT subsequently. VP proteins were detected with the antibody B1 (Progen) by overnight incubation at 4° C. in blocking buffer (1:500 dilution), subsequent washing and incubation with secondary antibody (anti-mouse IgG-HRP; 1:2500 in blocking buffer). Finally, the membrane was rinsed again and incubated with super signal pico west substrate (Pierce) for 5 min at RT. AAV capsid proteins are expressed as expected from the different VP expression vectors.
[0238]
[0239] The position of ORF2 and the encoded protein AAP is shown in relation to the position of translation start codons of the Cap proteins VP1, VP2 and VP3, as well as the EcoNI and BsiWI restriction sites (as given and described in more detail in
[0240]
[0241] The nucleotide sequence of ORF2 of AAV2 (NCBI entrée number NC_001401) from position to 3343 (including the tga stop codon), as well as the respective protein sequence of AAP obtained upon translation of ORF2 starting with the first nucleotide of ORF2 is given. marks the nucleotide position of the ATG start codon of VP3 which is underlined and given in bold. The predicted AAP translation initiation codon CTG coding for L (leucine) also is underlined and marked in bold.
[0242]
[0243]
[0244]
[0245]
[0246]
[0247]
[0248]
[0249] Same experimental setup as described in
[0250]
[0251] Same experimental setup as described in
[0252] Monoclonal antibody anti-AU1 for detection of AAP-AU1 or polyclonal anti-AAP serum for detection of AAP-AU1 or C-terminally truncated AAP (AAPtru).
[0253]
[0254] Derivates of pVP2N-gfp harbouring stop codons in ORF2 of the cap gene fragment were co-transfected with VP3 expression plasmid pCMV-VP3/2809 into 293-T cells.
[0255]
[0256]
[0257]
[0258]
[0259]
[0260]
[0261]
[0262]
[0263]
[0264]
[0265]
[0266]
[0267]
[0268]
[0269]
[0270]
[0271]
[0272]
[0273]
[0274] Alignment of predicted AAP protein sequences derived from ORF2 of the cap gene of different parvoviruses. Conserved amino acids that are 100% identical in at least 60% of aligned sequences are represented as lines in the lower row. Position of the predicted AAV2 AAP translation start is highlighted by a frame. Non-translated sequences upstream of the potential translation initiation codons are included as well. NCBI entrée numbers of the corresponding DNA sequences are listed in table 8.
[0275]
[0276] Virus-like particles assembled of VP1, VP2 and VP3 (VP1,2,3 VLP) or assembled only of VP3 (VP3 VLP) as indicated.
[0277]
[0278] Capsid formation in 293-T cells from constructs pCMV_VP3/2809 of AAV2 (AAV2-VP3), pCMV_AAV1VP3/2829 from AAV1 (AAV1-VP3) and a corresponding AAV5 VP3 construct (AAV5-VP3) co-transfected with pVP2N-gfp from AAV2, AAV1 and AAV5 as indicated was quantified by an ELISA based on monoclonal antibody A20. Bluescript vector (pBS) was used as negative control. Asterisks indicate samples for which no capsids could be detected.
AMINO ACID SEQUENCES
[0279]
TABLE-US-00006 SEQ ID NO: 1 ILVRLETQTQ YLTPSLSDSH QQPPLVWELI RWLQAVAHQW QTITRAPTEW VIPREIGIAI PHGWATESSP PAPEPGPCPP TTTTSTNKFP ANQEPRTTIT TLATAPLGGI LTSTDSTATF HHVTGKDSST TTGDSDPRDS TSSSLTFKSK RSRRMTVRRR LPITLPARFR CLLTRSTSSR TSSARRIKDA SRRSQQTSSW CHSMDTSP SEQ ID NO: 2 SSRHKSQTPP RASARQASSP LKRDSILVRL ATQSQSPIHN LSENLQQPPL LWDLLQWLQA VAHQWQTITK APTEWVMPQE IGIAIPHGWA TESSPPAPAP GPCPPTITTS TSKSPVLQRG PATTTTTSAT APPGGILIST DSTATFHHVT GSDSSTTIGD SGPRDSTSNS STSKSRRSRR MMASQPSLIT LPARFKSSRT RSTSFRTSSA LRTRAASLRS RRTCS SEQ ID NO: 3 ISVRLATQSQ SQTLNLSENH QQPPQVWDLI QWLQAVAHQW QTITRVPMEW VIPQEIGIAI PNGWATESSP PAPEPGPCPL TTTISTSKSP ANQELQTTTT TLATAPLGGI LTLTDSTATS HHVTGSDSLT TTGDSGPRNS ASSSSTSKLK RSRRTMARRL LPITLPARFK CLRTRSISSR TCSGRRTKAV SRRFQRTSSW SLSMDTSP SEQ ID NO: 4 LNPPSSPTPP RVSAKKASSR LKRSSFSKTK LEQATDPLRD QLPEPCLMTV RCVQQLAELQ SRADKVPMEW VMPRVIGIAI PPGLRATSRP PAPEPGSCPP TTTTSTSDSE RACSPTPTTD SPPPGDTLTS TASTATSHHV TGSDSSTTTG ACDPKPCGSK SSTSRSRRSR RRTARQRWLI TLPARFRSLR TRRTNCRT SEQ ID NO: 5 TTTFQKERRL GPKRTPSLPP RQTPKLDPAD PSSCKSQPNQ PQVWELIQCL REVAAHWATI TKVPMEWAMP REIGIAIPRG WGTESSPSPP EPGCCPATTT TSTERSKAAP STEATPTPTL DTAPPGGTLT LTASTATGAP ETGKDSSTTT GASDPGPSES KSSTFKSKRS RCRTPPPPSP TTSPPPSKCL RTTTTSCPTS SATGPRDACR PSLRRSLRCR STVTRR SEQ ID NO: 6 SSRHKSQTPP RALARQASSP LKRDSILVRL ATQSQSPTHN LSENLQQPPL LWDLLQWLQA VAHQWQTITK APTEWVMPQE IGIAIPHGWA TESSPPAPEH GPCPPITTTS TSKSPVLQRG PATTTTTSAT APPGGILIST DSTAISHHVT GSDSSTTIGD SGPRDSTSSS STSKSRRSRR MMASRPSLIT LPARFKSSRT RSTSCRTSSA LRTRAASLRS RRTCS SEQ ID NO: 7 SRHLSVPPTP PRASARKASS PPERDSISVR LATQSQSPTL NLSENLQQRP LVWDLVQWLQ AVAHQWQTIT KVPTEWVMPQ EIGIAIPHGW ATESLPPAPE PGPCPPTTTT STSKSPVKLQ VVPTTTPTSA TAPPGGILTL TDSTATSHHV TGSDSSTTTG DSGPRSCGSS SSTSRSRRSR RMTALRPSLI TLPARFRYSR TRNTSCRTSS ALRTRAACLR SRRTSS SEQ ID NO: 8 SHHPSVLQTP LRASARKANS PPEKDSILVR LATQSQFQTL NLSENLQQRP LVWDLIQWLQ AVAHQWQTIT KAPTEWVVPR EIGIAIPHGW ATESSPPAPE PGPCPPTTTT STSKSPTGHR EEPPTTTPTS ATAPPGGILT LTDSTATFHH VTGSDSSTTT GDSGPRDSAS SSSTSRSRRS RRMKAPRPSP ITSPAPSRCL RTRSTSCRTF SALPTRAACL RSRRTCS SEQ ID NO: 9 SSLLRNRTPP RVLANRVHSP LKRDSISVRL ATQSQSQTLN QSENLPQPPQ VWDLLQWLQV VAHQWQTITK VPMEWVVPRE IGIAIPNGWG TESSPPAPEP GPCPPTTITS TSKSPTAHLE DLQMTTPTSA TAPPGGILTS TDSTATSHHV TGSDSSTTTG DSGLSDSTSS SSTFRSKRLR TTMESRPSPI TLPARSRSSR TQTISSRTCS GRLTRAASRR SQRTFS SEQ ID NO: 10 TLGRLASQSQ SPTLNQSENH QQAPLVWDLV QWLQAVALQW QTITKAPTEW VVPQEIGIAI PHGWATESSP PAPEPGPCPP TTTTSTSKSP TGHREEAPTT TPTSATAPPG GILTSTDSTA TSHHVTGSDS STTTGDSGQK DSASSSSTSR SRRSRRMKAP RPSPITLPAR FRYLRTRNTS CRTSSAPRTR AACLRSRRMS S SEQ ID NO: 11 SHHKSPTPPR ASAKKANNQP ERGSTLKRTL EPETDPLKDQ IPAPCLQTLK CVQHRAEMLS MRDKVPMEWV MPRVIGIAIP PGLRARSQQP RPEPGSCPPT TTTCTCVSEQ HQAATPTTDS PPPGDILTST DSTVTSHHVT GKDSSTTTGD YDQKPCALKS SISKLRRSQR RTARLRSLIT LPARFRYLRT RRMSSRT SEQ ID NO: 12 KRLQIGRPTR TLGRPRPRKS KKTANQPTLL EGHSTLKTLE QETDPLRDHL PEKCLMMLRC VRRQAEMLSR RDKVPMEWVM PPVIGIAIPP GQRAESPPPA PEPGSYPRTT TTCTCESEQR PTATPTTDSP PPGDTLTLTA STATFPHATG SDSSTTTGDS GRNRCVLKSS TYRSRRSRRQ TARLRSLITL PARFRSLRIR RMNSHT SEQ ID NO: 13 SRVLKSQTPR AELARKANSL PERDSTLTTN LEPETGLPQK DHLPELCLLR LKCVQQLAEM VAMRDKVPRE WVMPPVIGIA IPLGQRATSP PPQPAPGSCR PTTTTCTCGS ARATPATPST DSPPPGDTLT LTASTATSRQ ETGKGSSTTT GDCAPKACKS ASSTSKLRRS RRLTGRRPYP TTSPARSRSL RTARTSSRT SEQ ID NO: 14 VKPSSRPKRG FSNPLVWWKT QRRLRPETSG KAKTNLVCPT LLHRLPRKTR SLARKDLPAG QKIRAKAPLP TLEQQHPPLV WDHLSWLKEV AAQWAMQARV PMEWAIPPEI GIAIPNGWKT ESSLEPPEPG SCPATTTTCT NESKDPAEAT TTTNSLDSAP PGDTLTTIDS TATFPRETGN DSSTTTGASV PKRCALDSLT SRLKRSRSKT STPPSATTSP VRSRSLRTRT TNCRTSSDRL PKAPSRRSQR ISTRSRSTGT AR SEQ ID NO: 15 ILVRLATQSQ SQTLNHSDNL PQPPLVWDLL QWLQAVAHQW QTITRVPMEW VIPQEIGIAI PNGWATESSP PAPAPGPCPP TTITSTSKSP ANQEPPTTTT TLATAPPGGI LTSTDSTATF HHVTGKDSST TTGDSDPRDS TSSSLTFKSK RSRRMTVRRR LPITLPARFR CLLTPSTSSR TSSARRIRDA SRRSQQTSSW SHSMDTSP SEQ ID NO: 16 TRRTVSSLPL QRRPKLEALP PPAIWDLVRW LEAVARQSTT ARMVPMEWAM PREIGIAIPH GWTTVSSPEP LGPGICQPTT TTSTNDSTER PPETKATSDS APPGDTLTST ASTVISPLET GKDSSTITGD SDQRAYGSKS LTFKLKKSRR KTQRRSSPIT LPARFRYLRT RSTSSRT SEQ ID NO: 17 LNNPTTRPGP GRSVPNASTT FSRKRRRPRP SKAKPLLKRA KTPEKEPLPT LDQAPPLVWD HLSWLKEVAV QWAMQAKVPT EWAIPREIGI AIPNGWTTES LPEPLEPGSC PATTTTCTSG SKDREEPTPT INSLDSAPPG GTLTTTDSTA TSPPETGNDS STTTGASDPK RCALDSLTSR LKKSLSKTPT PPSPTTSPAR SKSLRTRTTS CRTSSDRLQR APSRRSQRIS TRSRSMVTAR SEQ ID NO: 18 TTTFQKERRL GPKRTPSLPP RQTPKLDPAD PSSCKSQHNQ PQVWELIQCL REVAAHWATI TKVPMEWAMP REIGIAIPRG WGTESSPSPP APGCCPATTT TSTERSKAAP STEATPTPTL DTAPPGGTLT LTASTATGAP ETGKDSSTTI GASDPGLSES KSSTSKSKRS RCRTPPPPSP TTSPPPSKCL RTTTTNSRTS SATGPRDACR PSPRRSLRCR STATRR SEQ ID NO: 19 ASRSRSWLLQ SSVHTRPRKP QRTRRVSRDR IPGRRPRRGS SSPISLDLQQ TYLHPHNSPS LPQGFPVWFL VRCLQEEALQ WTMLNKVPTE WAMPREIGIA IPNGWATEFS PDPPGPGCCP ATTTTCTSRS QTPPACTASP GADTLATAPP GGTSTSIAST ATSRPETGSA SSITTGASDP RDCESNSSTS RSRRSRLLIR RPRSPTTSRA RSRSSQTTST SCRTSAATPP RDACRRSPRT SSRCRSTATR R SEQ ID NO: 20 KTEEPPRRAP NLWQHLKWQR EEAELWATLQ GVPMEWVMPR EIGIAIPNGW ETQSSQRPPE PGSCQATTTT STKQLPVEPL KMQMSSMQDT VPPGGTLIST ASTATSPLET GRDLSTTIGE SDPNLLNSRS SMSKSKKSQR RIKQRPLQTI SPQRFKSLRM MSINSRMSWA RLRKAPCRRS RRMSMPCRST GTAQCTPTRM EHGSMTVVHS TA SEQ ID NO: 21 KSLNYLKKTL LHPVIVEEKQ VQLPPKAPNL WQHLTWQREE AELWATLQGV PMEWVMPQEI GIAIPNGWET QSLPRLQEPG SCQATTTTST KPSQAEQTQT QIPNMLDTAP PGGTLISTDS TAISLQETGR DSSTTIGGLD RKHSNSRYSM CKLKKSRRKT RQRLLLTTLP LQSRYSRIMN TSCPMFWARP RRGRCHRSPQ MCMPCPSTAT AQCTPTRVEL DSMTEVPSIA SEQ ID NO: 22 TNTILKLKRP NKACRYQLHL KAEKKKLHRH NLEGAQQVPI LAAHLSWLQE EAVRWQTITR APREWVIPQV IGIAIPSGWE TTSLQSQPEL GCSPLTGIIS TGLSTLTAPQ VRVLMQPMQD TRLPGGTLTS IDSIATSPPE TGKDSSTTTQ ASGRKDSKSK SLTSKSKKLQ HKIQRKQLPT ISPAPYRSLR TRTTTYHMY SEQ ID NO: 143 LNNPTTRPGP GRSVPNASTT FSRKRRRPRP SKAKPLLKRA KTPEKEPLPT LDQAPPLVWD HLSWLKEVAV QWAMQAKVPT EWAIPREIGI AIPNGWTTES LPEPLEPGSC PATTTTCTSG SKDREEPTPT INSLDSAPPG GTLTTTDSTA TSPPETGNDS STTTGASDPK RCALDSLTSR LKKSLSKTPT PPSPTTSPAR SKSLRTRTTS CRTSSDRLQR APSRRSQRIS TRSRSMVTAR
EXAMPLES
[0280] The following examples exemplify the invention for AAV, especially for AAV2. Due to the general similarities within the structures of the adeno-associated viruses and other parvoviruses the invention can be easily transferred to other parvoviruses encoding 3 viral capsid proteins.
1. General Methods
1.1. Production of AAV (Like Particles) in Insect Cells
[0281] For production of AAV particles in Sf9 cells (cultivated in Graces (JHR Bioscience, USA)/10% FCS) cells were transfected with the vector plasmid pVL_VP1_MOD4, pVL_VP2 or pVL_VP3, derivates of the pVL1393 Polyhedrin Promoter-Based Baculovirus Transfer Vector (BD Bioscience, San Jose, Calif., USA) harboring a modified AAV VP1 open reading frame. (Cloning of pVL_VP1_MOD4, pVL_VP2 and pVL_VP3 is described in example 9)
[0282] Transfection was performed using the BaculoGold™ Transfection Kit according to manufacturer's manual (BD Bioscience, San Jose, Calif., USA). Following transfection cells were incubated at 27° C. 5 days after transfection the supernatant was used for single clone separation via an end point dilution assay (EPDA). For that purpose Sf9 cells were cultivated in well plates (2×10.sup.4 cells/well) and infected with serial dilutions of the transfection supernatant. 7 days after incubation at 27° C. the supernatant was transferred into a new 96 well plate (master plate) and stored at 2-8° C. The cells of the EPDA are lysed with sodium hydroxide, neutralized with sodium acetate and treated with Proteinase K. Following an Immune detection with the DIG-DNA wash and Block Buffer Kit (Roche, Mannheim, Germany) single clones could be detected.
[0283] To amplify single clones the according well from the master plate was used to infect Sf9 cells. Amplification of the recombinant Baculovirus was performed through several passages. Each passage was incubated for 3 days at 27° C. prior of use of the supernatant to infect cells for the next passage. In the first passage 1.2×10.sup.5 Sf9 cells (12 well plates) were infected with 50 μl of the supernatant out of the according well of the master plate. Supernatant was used to infect 2×10.sup.6 Sf9 (T25 Flask) (passage 1B). For passage 2,2×10.sup.7 Sf9 (T175 Flasks) were infected with 1 ml supernatant from passage 1B.
[0284] The virus titer of supernatant of passage 2 (P2) was analyzed via an end point dilution assay.
[0285] To produce AAV 1×10.sup.6/well Sf9 (6 well plates) were infected with supernatant of P2 with a multiplicity of infection (MOI) of 1. Cultures were incubated at 27° C. for 2-3 days. Cells were harvested and disrupted by a freeze and thaw process and analyzed for AAV production. AAV2 titer was analyzed using a commercially available AAV2 titration ELISA kit (Progen, Heidelberg, Germany) according to the manufacturer's manual.
1.2. Production of AAV (Like Particles) in Mammalian Cells
1.2.1. Plasmids
[0286] Ad Helper Plasmid
[0287] An Ad helper plasmid encoding adenoviral proteins E2, E4 and VAI-VAII was used for AAV manufacturing in 293 or 293-T cells. The helper plasmid pUCAdE2/E4-VAI-VAII was constructed by subcloning the BamHI restriction fragment encoding the adenovirus (Ad) E2 and E4-ORF6 from pAdEasy-1 (Stratagene, La Jolla, USA) into the BamHI site of pUC19 (Fermentas, St. Leon-Rot, Germany). The resulting plasmid is referred to as pUCAdE2/E4. The VAI-VAII fragment from pAdVAntage™ (Promega, Mannheim, Germany) was amplified by PCR using the primers
TABLE-US-00007 XbaI-VAI-780-3′: (SEQ ID NO: 59) 5′-TCT AGA GGG CAC TCT TCC GTG GTC TGG TGG-3′, and XbaI-VAII-1200-5′: (SEQ ID NO: 60) 5′-TCT AGA GCA AAA AAG GGG CTC GTC CCT GTT TCC-3′
[0288] cloned into pTOPO (Invitrogen, Carlsbad, USA) and then subcloned into the XbaI site of pUCAdE2/E4. This plasmid was named pUCAdV.
[0289] AAV Encoding Plasmids
[0290] The construction of pUCAV2 is described in detail in U.S. Pat. No. 6,846,665. Plasmid pTAV2.0 is described in (Heilbronn et al., 1990), pVP3 is described in (Warrington et al., 2004). Further AAV viral protein encoding plasmids are described within the respective examples.
1.2.2. Transfection for Large Scale Virus Production
[0291] 293-T cells (ATCC, Manassas, USA) (7.5×10.sup.6/dish) were seeded in 15 cm dishes (i.e. dish with a diameter of 15 cm) 24 h prior to transfection (cultivated in DMEM/10% FCS). Cells were transfected by calcium phosphate precipitation as described in US 2004/0053410.
[0292] In case of AAV promoter p40 dependent transcription a co-transfection with an adenoviral helper plasmid was performed. For co-transfection of the AAV encoding plasmid and pUCAdV a molar ratio of the plasmids of 1:1 was chosen. For transfection of one culture plate with 293-T cells the calcium phosphate transfection protocol was used as described above, 12 μg AAV Cap encoding plasmid (pUCAV2, pTAV2.0, and pVP3, respectively) and 24 μg pUCAdV were used. In case of p40 independent transcription cells were transfected with the respective AAV VP1, VP2 and/or VP3 encoding plasmid. For transfection of one culture plate of 293-T cells the calcium phosphate transfection protocol was used as disclosed in US 2004/0053410, 36 μg total DNA were mixed in 875 μl 270 mM CaCl.sub.2. In brief, 875 μl 2×BBS (50 mM BES (N,N-Bis-(2-hydroxyethyl)-2-aminoethane sulfonic acid) (pH 6.95), 280 mM NaCl and 1.5 mM Na.sub.2HPO.sub.4) was added to the mixture and the resulting solution was carefully mixed by pipetting. The solution was incubated for 20 min at room temperature (RT) and then added drop-wise to the cell culture plate. After 18 h incubation of cells in a humidified atmosphere at 35° C. and 3% CO.sub.2, medium was changed into a serum free DMEM (Invitrogen Carlsbad, USA) and cells were cultivated for an additional 3 d at 37° C., 5% CO.sub.2 in a humidified atmosphere.
[0293] 293-T cells were harvested with a cell lifter, transferred into 50 ml plastic tubes (Falcon) and centrifuged at 3000 g, 4° C. for 10 min. The cell pellet was resuspended in 0.5 ml lysis buffer (150 mM NaCl, 50 mM Tris, pH 8.5) per 15 cm dish and objected to three rounds of freeze and thaw cycles (liquid nitrogen/37° C.). The cell lysate was cleared by two centrifugation steps (3700 g, 4° C., 20 min) and the AAV-containing supernatant was used for further purification. Alternatively the whole dishes were objected to freeze and thaw cycles (−50° C./RT). The remaining supernatant was collected and further purified as described in 1.3.
1.2.3. Small Scale Transfection and Preparation of Virus Supernatants
[0294] Cells (5×10.sup.5/dish) were seeded in 6 cm dishes 24 h prior to transfection. 293-T cells were transfected by calcium phosphate precipitation as described in US 2004/0053410. For HeLa and COS-1 cells transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, USA) according to the manufacturer's manual. In case of promoter p40 dependent transcription of the cap gene (pTAV2.0, derivates thereof, and pVP3) cells were infected with adenovirus type 5 (Ad5) (MOI=10). After additional incubation for 24-48 h, cells were harvested in the medium and lysed by three freeze-thaw cycles (−80° C. and 37° C.). Lysates were incubated at 56° C. for 30 min to inactivate Ad5. Cell debris was removed by centrifugation at 10000 g for 5 min.
1.2.4. Cell Culture
[0295] HeLa and 293-T cells were maintained at 37° C. and 5% CO.sub.2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine.
1.3. Purification
1.3.1 Tangential Cross Flow Filtration (TFF) and Benzonase Treatment
[0296] After harvest the cleared cell culture medium was further concentrated using a Tangential Cross Flow Filtration Unit (Sartoflow Slice 200 Benchtop Crossflow System, Sartorius Biotech GmbH, Gottingen, Germany) using a 100 kDa cut off membrane (SARTOCON Slice 200). The resulting TFF concentrate was pooled with the supernatant (obtained as described in 1.2) and immediately treated with 100 U/ml benzonase (Merck, Darmstadt, Germany) at 37° C. for 2 h. After benzonase treatment the cell lysate was cleared by centrifugation at 3700 g, 4° C. for 20 min. Cleared supernatant was purified using size exclusion chromatography (ÅKTA explorer system, GE Healthcare, Munich, Germany).
1.3.2 Size Exclusion Chromatography (SEC)
[0297] Cleared supernatant was separated through a Superdex 200 (prep grade) packed XK 50 chromatography column (250 mm in height and 50 mm in diameter; GE Healthcare, Munich, Germany). SEC fractions (5 ml each) were collected and the capsid titer was determined using the AAV2 capsid-specific A20 ELISA (Progen, Heidelberg, Germany, Cat. No: PRATV). SEC fractions containing AAV2 particles were pooled and further purified using iodixanol- or sucrose-density ultracentrifugation.
(i) Purification of AAV Particles by Density Gradient Centrifugation Using Iodixanol
[0298] The virus-containing SEC pool was transferred to Qickseal ultracentrifugation tubes (26×77 mm, Beckman Coulter, Marseille, France). Iodixanol solutions (purchased from Sigma, Deisenhofen, Germany) of different concentrations were layered beneath the virus containing lysate. By this an Iodixanol gradient was created composed of 6 ml 60% on the bottom, 5 ml 40%, 6 ml 25% and 9 ml 15% Iodixanol with the virus solution on top. The gradient was spun in an ultracentrifuge at 416000 g for 1 h at 18° C. The 40% phase containing the AAV particles was then extracted with a canula by puncturing the tube underneath the 40% phase and allowing the solution to drip into a collecting tube until the 25% phase was reached.
(ii) Sucrose Density Gradient Analysis
[0299] 1.5×10.sup.6 cells were seeded in 10 cm dishes 24 h prior to transfection. They were harvested 48 h post transfection and lysed in 300 μl PBS-MK (phosphate-buffered saline: 18.4 mM Na.sub.2HPO.sub.4, 10.9 mM KH.sub.2PO.sub.4, 125 mM NaCl supplemented with 1 mM MgCl.sub.2, 2.5 mM KCl) by five freeze-thaw cycles (−80° C. and 37° C.). After treatment with 50 U/ml Benzonase (Sigma, Deisenhofen, Germany) for 30 min at 37° C. and centrifugation at 3700 g for 20 min the supernatant was loaded onto a 11 ml 5-30% or 10-30% sucrose gradient (sucrose in PBS-MK, 10 mM EDTA, containing one tablet of complete mini EDTA free protease inhibitor (Roche, Mannheim, Germany)) in polyallomer centrifuge tubes (14 by 89 mm; Beckman Coulter, Marseille, France). After centrifugation at 160000 g for 2 h at 4° C. (SW41 rotor; Beckman), 500 μl fractions were collected from the bottom of the tubes. As reference empty AAV2 capsids (60 S) were analyzed in a separate gradient. For immuno dot blot assay 50 μl of heat denatured (99° C. for 10 min) or non denatured aliquots of the fractions were transferred to Protran nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) using a vacuum blotter. Membranes were blocked for 1 h in PBS containing 10% skim milk powder and then incubated for 1 h with monoclonal antibodies B1 (Progen, Heidelberg, Germany, Cat. No: 65158) to detect denatured capsid proteins or A20 to detect non denatured capsids. Antibodies B1 and A20 were applied in 1:10 dilutions. Membranes were washed several times with PBS and incubated for 1 h with a peroxidase-coupled goat anti-mouse antibody (1:5000 dilution) (Dianova, Hamburg, Germany). Then, membranes were washed again and the antibody reaction was visualized using an enhanced chemiluminescence detection kit (Amersham, Braunschweig, Germany). For Western blot analysis 15 μl per fraction were processed for SDS-PAGE and then probed with monoclonal antibodies A69 (Progen, Heidelberg, Germany, Cat. No: 65157) or B1.
(iii) Purification of AAV Particles by Chromatography
Purification of Empty wtVP3# and Modified AAVLPs*
[0300] Indices # and * refer to slight differences in the purification protocol between wtAAV # and modified AAVLPs*. Buffer ingredients are marked correspondingly.
Cation Exchange Chromatography (ÅKTA Explorer System)
[0301] Total lysate containing empty wtVP3# and modified AAVLPs* was obtained by performing three freeze thaw cycles (−54° C./37° C.). Total lysate was cleared by centrifugation at 4100 rpm, 4° C., min (MULTIFUGE L-R; Heraeus, Hanau, Germany). The pH of the resulting cleared supernatant was adjusted to 6. In addition, the conductivity of salt was reduced to approximately 10 mS/cm by adding sterile water.
[0302] A Fractogel EMD SO.sub.3.sup.− (M) chromatography column (100 mm in height; 15 mm in diameter, XK16, GE Healthcare, München, Germany) was packed and equilibrated using 5 CV running buffer consisting of 80 mM NaCl, 2% sucrose, 50 mM HEPES (pH 6.0), 2.5 mM MgCl.sub.2.
[0303] After equilibration, cleared supernatant was separated through the Fractogel EMD SO.sub.3.sup.− (M) packed chromatography column (flow rate 10 ml/min). After separation, column was washed using 5 CV running buffer mentioned above. Bound particles (wtVP3 or modified AAVLPs) were effectively eluted at a sodium chloride concentration of 350 mM (peak 1=45 ml).
Buffer Exchange (ÅKTA Explorer System)
[0304] To adjust the pH and the salt concentration of the eluted proteins (peak 1) for successive anion exchange chromatography, buffer exchange was performed using a Sephadex G25 packed chromatography column (500 mm in height; 15 mm in diameter, XK26, GE Healthcare, München, Germany) (flow rate 10 ml/min). After column equilibration using 3 CV SOURCE 15Q running buffer consisting of 25 mM Tris (pH 8.2), 150 mM NaCl#/100 mM NaCl*, 2.5 mM MgCl.sub.2 peak 1 was separated through the column. Protein fraction (=120 ml) was collected.
Anion Exchange Chromatography (ÅKTA Explorer System)
[0305] A SOURCE 15Q chromatography column (80 mm in height; 15 mm in diameter, XK16, GE Healthcare, München, Germany) was equilibrated using 5 CV SOURCE 15Q running buffer consisting of 25 mM Tris (pH 8.2), 150 mM NaCl#/100 mM NaCl*, 2.5 mM MgCl.sub.2. After equilibration, the protein fraction obtained after buffer exchange (appr. 120 ml) was loaded and separated through the chromatography column (flow rate 10 ml/min). Flow-through containing 90% of the particles (appr. 120 ml) was collected.
Particle Concentration Using Centrifugal Filter Devices
[0306] Flow-through containing wtVP3# or modified AAVLPs* was concentrated using Centricon Plus-(cut off 100 kDa) centrifugal filter devices (Millipore). Concentration was carried out using a swinging-bucket rotor (MULIFUGE L-R; Heraeus, Hanau, Germany) at 3500 g, 20° C. for 15 min. Resulting concentrate (appr. 45 ml) was immediately separated through a size exclusion chromatography.
Size Exclusion Chromatography (ÅKTA Explorer System)
[0307] A Superdex 200 (prep grade) chromatography column (500 mm in height; 50 mm in diameter, XK50, GE Healthcare, München, Germany) was packed and equilibrated using 2 CV running buffer consisting of 200 mM NaCl, 2% sucrose, 50 mM HEPES (pH 6.0), 2.5 mM MgCl.sub.2. The concentrate mentioned above (appr. 45 ml) was separated through the column (fow-rate 10 ml/min). Particles eluted first (SEC fraction no. 1-13; each 5 ml). SEC fractions with a particle purity of greater than 95% were pooled, sterile filtered (0.2 μm) (Minisart; Sartoriusstedim) and stored at −84° C.
1.4. Analysis of Protein Expression by Western Blot
[0308] Identical portions of harvested cells or identical amounts of purified particles were processed for SDS-PAGE. Protein expression was analyzed by Western blot assay using monoclonal antibodies A69, B1 (Progen, Heidelberg, Germany), anti-AU1 (Covance, Emeryville, USA), anti-GFP (clone B-2; Santa Cruz Biotechnology, Santa Cruz, USA) or polyclonal antibody anti-AAP (see 1.7.) as described previously (Wistuba et al., 1995). Variations of the protocols are indicated within the description of the respective examples.
1.5. Titer Analysis
[0309] Capsid titers were determined using a commercially available AAV2 titration ELISA kit (Progen, Heidelberg, Germany Cat. No: PRATV) or the respective AAV1 titration ELISA kit (Progen, Heidelberg, Germany Cat. No: PRAAV1) according to the manufacturer's manual.
1.6. Immunofluorescence Analysis
[0310] For immunofluorescence analysis HeLa cells were cultivated for 24 h on coverslips, transfected and in case of promoter p40 dependent transcription of the cap gene (pTAV2.0 and pVP3) infected with Ad5 (MOI=4). After 20-48 h cells were fixed with 100% methanol (10 min, −20° C.) and washed with PBS (phosphate-buffered saline: 18.4 mM Na.sub.2HPO.sub.4, 10.9 mM KH.sub.2PO.sub.4, 125 mM NaCl). Incubation with primary antibodies was performed for 1 h at RT or over night at 4° C. As primary antibodies hybridoma supernatants A20 or A69 were used to detect assembled capsids or VP2 respectively. A20 and A69 were used undiluted (Progen, Heidelberg, Germany). For detection of unassembled capsids a rabbit polyclonal serum was used in a 1:500 dilution to label all three free VP proteins. Coverslips were washed three times with PBS and thereafter incubated with appropriate secondary antibodies (Cy 3 labeled goat anti mouse in 1:400 dilution or FITC labeled goat anti rabbit 1:150 purchased from Dianova, Hamburg, Germany or Molecular Probes, Leiden, The Netherlands) for 1 h at RT. Coverslips were washed again, dipped into 100% ethanol and embedded in Permafluor mounting medium (Beckman Coulter, Marseille, France). Confocal images (0.3 μm sections) were obtained with a Leica TCS SP2 laser scanning microscope and further processed using Adobe Photoshop CS software. Variations of the protocols are indicated within the description of the respective examples.
[0311] To visualize GFP expression, cells were fixed with 2% paraformaldehyde for 15 min, quenched twice with 50 mM NH.sub.4Cl for 5 min, and permeabilized with 0.2% Triton X-100 for 10 min.
1.7. Preparation of Polyclonal Antibody
[0312] The polyclonal AAP antiserum (anti-AAP) was generated by immunization of a guinea pig with a peptide comprising the sequence GKDSSTTTGDSDPRDSTS (SEQ ID NO: 61) conjugated to KLH (Keyhole Limpet Hemocyanin) following standard procedures.
1.8. Negative Staining of Virus Particles for Electron Microscopy
[0313] For electron microscopy according to (Grimm et al., 1999, Grimm and Kleinschmidt, 1999, Mittereder et al., 1996), negative staining of virus particles was performed as described in detail below.
[0314] Five μl of sample (about 5×10.sup.10 virus particles) were applied onto the freshly air-glow discharged carbon coated side of a grid and incubated for 2 min. Excess solution was removed by blotting the edge of the grid onto Whatman filter paper. To avoid salt precipitates, the grid was washed with 3 drops of water followed by four drops of 2% (w/v) uranyl acetate solution. The last droplet of staining solution was allowed to sit on the grid for 5 min before blotting and air drying. Electron micrographs were taken with a Morgagni 268D FEI microscope at 100 kV.
2. Analysis of VLP Formation by N-Terminal Deletion Analysis of VP2
[0315] Our as well as previous studies (compare above) reported a lack of capsid assembly when VP3 is expressed from constructs comprising the cds of VP3 alone. Since expression of VP3 is not sufficient for VLP formation, we tried to identify further sequences which could overcome this defect. In this experiment we checked whether a sequence upstream of the VP3 cds was necessary for VLP formation. If yes, the sequence should be characterized.
2.1. Cloning of Deletion Mutants
[0316] Plasmids pTAV2.0 (Heilbronn et al., 1990), pVP3 (Warrington et al., 2004), pCMV-VP (Wistuba et al., 1997) and pKEX-VP3 (Ruffing et al., 1992) have been described previously. The deletion mutants pCMV-VP3/1882, pCMV-VP3/2193, pCMV-VP3/2596, pCMV-VP3/2611, pCMV-VP3/2696, pCMV-VP3/2765 and pCMV-VP3/2809 were cloned from plasmid pVP3. Numbers behind the name of the pCMV-VP3 plasmid indicate the nucleotide position in the AAV2 genome according to Ruffing et al. (1994). Constructs are schematically shown in
[0317] For cloning of deletion mutants, the HindIII/BsiWI fragment of pVP3 (with mutated VP1 and VP2 translation start codons) was subcloned into the HindIII/BsiWI backbone of pCMV-VP resulting in the construct pCMV-VP3/1882 (
TABLE-US-00008 (SEQ ID NO: 62) 5′-CCTCTGGTCTGGGAACTAAGCTTATGGCTACAGGCAGTGGCG-3′ (SEQ ID NO: 63) 5′-CGCCACTGCCTGTAGCCATAAGCTTAGTTCCCAGACCAGAGG-3′
[0318] HindIII/BsiWI fragments from mutated plasmids were then subcloned into the HindIII/BsiWI backbone of pCMV-VP resulting in constructs pCMV-VP3/2611, pCMV-VP3/2696, pCMV-VP3/2765 and pCMV-VP3/2809 (
2.2. Analyses of Constructs by Western Blot and ELISA
[0319] For analysis of protein expression identical portions of harvested cells were processed for SDS-PAGE.
[0320] As shown in
[0321] When, however, extracts of cells transfected with pVP3 were probed with antibody A69 which detects only VP1 and VP2, thus omitting the reaction with the abundant VP3, one could detect faint bands in the region of VP1 and VP2 which were absent in extracts of cells transfected with pKEX-VP3. This result suggests that transfection of the pVP3 construct leads to the expression of small amounts of VP1 and VP2 or VP1- and VP2-like proteins. They are possibly translated from alternative translation initiation codons or by unscheduled initiation at the mutated VP1 and VP2 translation initiation sites.
[0322] Antibody A69 revealed in all deletion mutants of pVP3 up to pCMV-VP3/2696 one or several polypeptide band(s), only Western blots with extracts of cells transfected with pCMV-VP3/2765 and pCMV-VP3/2809 showed no reaction with A69 because the antibody epitope was already deleted in these proteins.
[0323] Capsid assembly was confirmed by an antibody A20 based capsid ELISA (
[0324] In agreement with our previous results, expression of VP3 alone by transfecting pCMV-VP3/2809—which is equivalent to pKEX-VP3—did not lead to detectable capsid formation (
2.3 Conclusion
[0325] This result shows a clear correlation between the presence of N-terminally extended VP3 sequence (due to the presence of DNA sequence upstream of the VP3 start codon) and capsid assembly. We identified a DNA sequence of about 44 nucleotides upstream of the VP3 cds that has to be present in addition to the VP3 cds for VP3 VLP formation. This 44 nt confers to construct pCMV-VP3/2765 which still is able to cause capsid assembly.
[0326] The presence of some more DNA sequence upstream of the 2765′ site increases efficiency of capsid assembly which is in line with ORF2 starting at nucleotide position 2717 and the putative start of the full-length AAP possibly located between nucleotide 2717 and 2765.
3. Sequence Fragment of the Cap Gene is Able to Induce Capsid Assembly in Trans
[0327] In example 2, we identified some sequence upstream of the VP3 start codon (comprised by fragment Z) that has to be present in addition to the VP3 cds for particle formation. To prove the hypothesis that the product of fragment Z functions transiently and in trans, we tested whether a capsid sequence fragment comprising the EcoNI/BsiWI restriction fragment fused to the cds of GFP can rescue the capsid assembly deficiency of VP3.
3.1. Cloning of pVP2N-gfp for Trans-Complementation
[0328] For generation of construct pVP2N-gfp, EcoNI and BsiWI restriction sites were introduced into the multiple cloning site of the vector pEGFP-N1 (BD Biosciences, Erembodegem, Belgium). Afterwards the EcoNI/BsiWI fragment from pTAV2.0 (position of restriction sites is given in
[0329] A number of derivates containing e.g. codon modifications or stop codons originated from pVP2N-gfp as schematically indicated in the respective figures. They always include the GFP cds and were named accordingly (with the addition −gfp). To simplify matters this appendix (−gfp) is missing to names of the respective constructs in some figures (e.g.
3.2. Analysis of Functional Substitution in Trans
[0330] The following experiments were performed in HeLa cells. Plasmids pCMV-VP3/2809 and pVP2N-gfp were co-transfected in different molar ratios and analyzed for gene expression and capsid assembly (
3.3. Conclusion
[0331] This result shows that presence of an EcoNI-BsiWI restriction fragment of the cap gene in trans rescues capsid assembly of constructs expressing VP3 as only capsid protein. Since assembly could be detected even at a 50-fold reduced amount of pVP2N-gfp plasmid co-transfected, a substoichiometric action of the helper factor for VP3 capsid assembly can be assumed.
4. C-Terminally Truncated VP2 Proteins are Expressed in Substoichometric Amounts and Become Incorporated into Capsids
[0332] Here it was investigated if the generated AAV like particles consist of VP3 only. Empty particles were produced from plasmid pCMV-VP3/2696 or in a trans-complementation assay of cotransfection of pCMV-VP/2809 and pVP2N-GFP Particles were purified via sucrose cushion according to Steinbach et al. (1997) with modification described by Kronenberg et al. (2001) and with the modification that the 293 cells were transfected without adenoviral infection and cells were harvested after 48 h. Incorporation of truncated VP2 protein was analyzed by Western blot (
[0333] pVP2N-GFP could not be detected within maximal loading of 5×10.sup.11 particles. But transfecting pCMV-VP3/2696 an A69 signal was detected which shows that a truncated VP2 is incorporated into the capsids substoichiometrically.
4.1. Result
[0334] In conclusion VP3 only particles are generated within the trans situation. In contrast in the cis situation a truncated VP2 is incorporated substochiometrically. From Western blot the signal intensity of VP1 from 2×10.sup.9 wt AAV particles is about the same as the signal from 1×10.sup.11 particles generated from pCMV-VP3/2696. This means the amount of truncated VP2 is about fold lower than the amount of VP1. Assuming a stoichiometric ratio of VP1:VP2:VP3 of 1:1:10 within a wt capsid there is approximately 500-fold less truncated VP2 than VP3. Since one capsid is composed of 60 VP subunits also capsids must exist that are composed of VP3 only.
4.2. Conclusion
[0335] This result strengthens the conclusion that the truncated VP2 protein itself is not required for the capsid itself.
5. Codon Modification of the Construct Used for Trans-Complementation can Inhibit the Trans-Complementation Process
[0336] To investigate the nature of the trans-complementing agent of the fragment Z, the VP2N part (part between restriction sites EcoNI and BsiWI) within pVP2N-gfp was codon modified. That means the DNA sequence was altered without changing the amino acid sequence of the first ORF. Codon modification was performed by GENEART (Regensburg, Germany). Codons were modified for codons preferentially used in mammalian cells. Sequence is shown in
[0337] Protein expression of pVP2Ncm-gfp was compared in Western blot analysis (
Result and Conclusion
[0338] Western blot showed that the protein expression from the codon modified construct (pVP2Ncm-gfp) was even higher than protein expression from the non-modified construct (pVP2N-gfp), not unexpected since the codon modification was optimized for mammalian cells (
[0339] To exclude a negative effect of the large amounts of pVP2Ncm-gfp protein on capsid assembly, we co-transfected the codon modified pVP2Ncm-gfp with pCMV-VP3/2696. In this combination capsid assembly was normal, showing that the assembly activity was not suppressed by the high amount of pVP2Ncm-gfp (data not shown). Also expression of lower amounts of pVP2Ncm-gfp by transfection of reduced amounts of plasmid pVP2Ncm-gfp together with pCMV-VP3/2809 did not rescue capsid assembly (data not shown).
[0340] This result strengthens the hypothesis that no protein translated from the first ORF is responsible for the trans-complementing activity.
6. Insertion of Stop Codons into the Construct Used for Trans-Complementation does not Inhibit the Trans-Complementation Process
[0341] To further analyze the nature of the trans-complementing agent stop codons were inserted within the EcoNI-BsiWI restriction fragment. To insert Stop codons point mutations were performed.
6.1. Insertion of Stop Codons into pVP2N-gfp
[0342] For construction of pVP2N/stopA-gfp (also named pVP2N/ORF1stopA-gfp), pVP2N/stopB-gfp (identical to pVP2N/ORF1 stopB-gfp), pVP2N/stopC-gfp (also named pVP2N/ORF1 stopC-gfp) and pVP2N/stopD-gfp (identical to pVP2N/ORF1 stopD-gfp) site-directed mutagenesis reactions (QuickChange site-directed mutagenesis kit, Stratagene) were performed using template pVP2N-gfp and two complementary PCR primers which included the desired substitutions. In each case the EcoNI/BsiWI fragment was then cloned into the EcoNI/BsiWI backbone of pVP2N-gfp.
[0343] For generation of StopA cytosine at nucleotide position 2770 were substituted to thymine resulting in a tag stop codon. For generation of StopB adenine at nucleotide position 2797 was substituted to thymine resulting in a tga stop codon. Stop C was generated by substituting adenine at nucleotide position 2821 to thymine and thymine at position 2823 to adenine, resulting in a tga stop codon. Stop D was created by substituting guanine at nucleotide position to thymine resulting in a tga stop codon. Positions are according to Ruffing et al. (1994).
TABLE-US-00009 Primer pairs used for insertion of Stop codons at four different sites within the pVP2N-gfp StopA 5′-CCA GCC TCT CGG ATA GCC ACC AGC AGC C-3′ (SEQ ID NO: 64) i-StopA 5′-GGC TGC TGG TGG CTA TCC GAG AGG CTG G-3′ (SEQ ID NO: 65) StopB 5′-GCC CCC TCT GGT CTG TGA ACT AAT ACG ATG GC-3′ (SEQ ID NO: 66) i-StopB 5′-GCC ATC GTA TTA GTT CAC AGA CCA GAG GGG GC-3′ (SEQ ID NO: 67) StopC 5′-CGA TGG CTA CAG GCT GAG GCG CAC CAA TGG C-3′ (SEQ ID NO: 68) i-StopC 5′-GCC ATT GGT GCG CCT CAG CCT GTA GCC ATC G-3′ (SEQ ID NO: 69) StopD 5′-GGA GTG GGT AAT TCC TCG TGA AAT TGG CAT TGC G-3′ (SEQ ID NO: 70) i-StopD 5′-CGC AAT GCC AAT TTC ACG AGG AAT TAC CCA CTC C-3′ (SEQ ID NO: 71)
[0344] Schematic presentation of the inserted stop codons is depicted in
[0345] Further protein expression of the Stop constructs was tested by Western blot analysis using the A69 antibody.
6.2. Result and Conclusion
[0346] Western blot analysis confirmed that VP3 is expressed in all samples (detected by monoclonal antibody B1 in
7. The Postulated NLS is not Necessary for VLP Formation
[0347] While mutant pCMV-VP3/2696 formed high capsid levels, the slightly shorter mutant pCMV-VP3/2765 assembled to clearly reduced amounts of capsids (
TABLE-US-00010 BC3-ala forward: (SEQ ID NO: 72) 5′-GGC GGG CCA GCA GCC TGC AGC AGC AGC ATT GAA TTT TGG TCA GAC TGG-3′ BC3-ala reverse: (SEQ ID NO: 73) 5′-CCA GTC TGA CCA AAA TTC AAT GCT GCT GCT GCA GGC TGC TGG CCC GCC-3′
[0348] Immunofluorescence of transfected HeLa cells with the A20 antibody (
[0349] This supports the interpretation that the sequence element comprising RKR168-170 does not act as a NLS in this context and might play a different role in capsid assembly.
8. Nuclear Localization (and N Terminal Extension) of VP Proteins is not Sufficient for Capsid Assembly
[0350] It has been reported that fusion of an NLS derived from the SV40 large T antigen to VP3 translocates VP3 into the nucleus and leads to capsid assembly (Hoque et al., 1999a). We repeated this experiment and observed efficient nuclear accumulation of VP3 protein, however, there was no capsid assembly detectable with antibody A20 (
[0351] Further, a heterologous N terminal extension upstream of VP3 (HSA) was tested to restore assembly competence to VP3.
[0352] Further several constructs were transfected in 293 cells to compare protein expression and assembly efficiency.
8.1. Cloning of Constructs
[0353] pCI-VP, pCI-VP2 and pCI-VP3 were cloned by PCR amplification of the respective VP coding regions using primer with XhoI (5′-) and NotI (3′-) overhangs and subcloning of the XhoI-/NotI-digested PCR products into the XhoI-/NotI-digested vector pCI (PROMEGA). In case of pCI-VP2, the start codon for VP2 was changed from ACG to ATG at the same time
[0354] Cloning of the construct pCMV-NLS-VP3 was carried out by site-directed mutagenesis reaction using the construct pCMV-VP3/2809 as template and the complementary PCR primers
TABLE-US-00011 (SEQ ID NO: 74) 5′-GGAAT TCGAT ATCAA GCTTG CCATG GCACC ACCAA AGAAG AAGCG AAAGG TTATG GCTAC AGGCA GTGG-3′ and (SEQ ID NO: 75) 5′-CCACT GCCTG TAGCC ATAAC CTTTC GCTTC TTCTT TGGTG GTGCC ATGGC AAGCT TGATA TCGAA TTCC-3′.
[0355] Then the HindIII/BsiWI fragment was subcloned from the amplicon into the HindIII/BsiWI backbone of pCMV-VP3/2809. The cap gene product NLS-VP3 contains the amino acid sequence of SV40 NLS MAPPKKKRKV at the N-terminus of VP3.
[0356] The construct pCMV-HSA-VP3 is also based on pCMV-VP3/2809 and contains a nucleic acid sequence coding for amino acids 25-58 of human serum albumin (HSA) directly upstream of the VP3 cds. Fragment
TABLE-US-00012 (SEQ ID NO: 76) 5′-GGTAC CAAGC TTACG GACGC CCACA AGAGC GAGGT GGCCC ACCGG TTCAA GGACC TGGGC GAGGA AAACT TCAAG GCCCT GGTGC TGATC GCCTT CGCCC AGTAC CTGCA GCAGT GCAAG CTTGA GCTC-3′
(with a HindIII restriction site at both ends) was obtained via gene synthesis (Geneart, Regensburg, Germany). After HindIII digestion of the corresponding vector the resulting 111 bp fragment was subcloned into the HindIII linearized pCMV-VP3/2809 backbone. Translation of VP3 is initiated at a standard ATG start codon whereas translation of HSA-VP3 (with 37 Aas elongation at VP3 N-terminus) is initiated at an ACG start codon.
8.2. Analyses of Constructs by Immunofluorescence and Sucrose Gradient
[0357] We transfected HeLa cells with the different constructs: pCMV-NLS-VP3 or pCMV-VP3/2809 either alone or in a co-transfection with pVP2N-GFP. Further pCMV-HSA-VP3 was transfected. Expression of capsid proteins and formation of capsids was analyzed by immunofluorescence as described above using a polyclonal VP antiserum or the monoclonal A20 antibody. Further capsid formation was analyzed within following a sucrose gradient.
Results
[0358] Just as Hoque et al. (1999a) and comparable to the wildtype (wt) and the proteins expressed from the N-terminally truncated construct pCMV/2696, we could express VP3 from the construct pCMV-NLS-VP3 and observed efficient nuclear accumulation of VP3 protein. However, in contrast to the wt and the N-terminally truncated construct pCMV/2696 we could not detect capsid assembly using the antibody A20 (
[0359] As expected, expression of the VP3 protein with a prolonged N-terminus consisting of 36 AA of human serum albumin (HSA-VP3), equivalent in length to the VP3 N-terminal extension of mutant pCMV-VP3/2696 could be detected by antibody staining (
[0360] Co-transfection of pVP2N-gfp induced capsid assembly, readily detectable by antibody A20 (
[0361] Analysis of possible assembly products—not reacting with the A20 antibody—by sucrose density gradient sedimentation showed very low amounts of VP protein containing material (sedimenting over the whole range of the gradient) which reacted with antibody B1 (
8.3. Analyses of Constructs by Western Blot and ELISA
[0362] A set of different constructs was analyzed for gene expression in Western Blot and in ELISA for capsid assembly (
Results
[0369] Western Blot analysis showed similar capsid protein expression of the different constructs with the expected size of the VP proteins (
Conclusion
[0370] The results show that nuclear accumulation of VP3 alone is not sufficient for capsid assembly and that a heterologous N-terminal extension upstream of VP3 is not able to bring about assembly competence to VP3.
[0371] Further our favored constructs pCI-VP2mutACG or pCMV-VP3/2696 lead to more than 3 log higher VP3 particle titers when compared to the NLS-VP3 fusion construct described by Hoque et al. (1999a). These experiments also demonstrate that VP3 N-terminal fusion constructs can assemble into VLPs. Therefore I-203 is a suitable insertion site for foreign peptide sequences.
9. VP3 Capsid Assembly can be Achieved in Insect Cells
9.1. Cloning of the VP1 Mutant “Modification 4”
[0372] The construct pVL_VP1_MOD4 was generated to produce viral particles consisting essentially of the capsid protein VP3 in the absence of any Rep expression.
[0373] In detail, pUC19AV2 (described in detail in U.S. Pat. No. 6,846,665) was used as template to amplify VP1 according to standard PCR conditions in the presence of the following primers:
TABLE-US-00013 Insect_mod_4_s: (SEQ ID NO: 77) 5′-CAC CCG CGG GGA TCC GCC GCT GCC GAC GGT TAT CTA CCC GAT TGG CTC-3′, and E_VP2_rev: (SEQ ID NO: 78) 5′-CGC GAA TTC CTA TTA CAG ATT ACG AGT CAG G-3′
[0374] Thereby, the wildtype translation start codon ATG (coding for Methionin) of VP1 was changed into GCC (Alanin) and inactivated. The resulting EcoRI/BamHI fragment was cloned into pBSIIKS (Stratagene, La Jolla, Calif., USA). This vector was used to inactivate the translation start codon of VP2 by site directed mutagenesis according to the instructions of the QuickChange II Site directed mutagenesis kit (Stratagene) using the following primers:
TABLE-US-00014 Insect-muta_4_s: (SEQ ID NO: 79) 5′-ACC TGT TAA GAC AGC TCC GGG AAA AAA G-3′ Insect-muta_4_as: (SEQ ID NO: 80) 5′-CTT TTT TCC CGG AGC TGT CTT AAC AGG T-3′
[0375] Thereby, the wildtype translation start codon ACG of VP2 was changed into ACA (both coding for Threonin). The resulting construct was digested with restriction enzymes BamHI and EcoRI and cloned into the baculo transfer vector pVL1393. As a result, the construct contained the complete AAV cap gene with mutations of the VP1 and VP2 start codons but no rep cds. (
9.2. Cloning of pVL_VP2
[0376] AAV2 VP2 was amplified using the primers E_VP2_for and E_VP2_rev listed below. Thereby, the wildtype VP2 translation start codon ACG (coding for Threonine) was changed into ATG (Methionine). Primers:
TABLE-US-00015 E_VP2_for: (SEQ ID NO: 81) 5′-CAC CCG CGG GGA TCC ACT ATGGCT CCG GGA AAA AAG AGG-3′ E_VP2_rev: (SEQ ID NO: 82) 5′-CGC GAA TTC CTA TTA CAG ATT ACG AGT CAG G-3′
[0377] The resulting construct was cloned into the baculo transfer vector pVL1393.
9.3. Cloning of pVL_VP3
[0378] AAV2 VP3 was amplified using the primers E_VP3_for and E_VP3_rev listed below. Primers:
TABLE-US-00016 E_VP3_for: (SEQ ID NO: 83) 5′-CAC CCG CGG GGA TCC ACT ATG GCT ACA GGC AGT GGC GCA C-3′ E_VP2_rev: (SEQ ID NO: 84) 5′-CGC GAA TTC CTA TTA CAG ATT ACG AGT CAG G-3′
[0379] The resulting construct was cloned into the baculo transfer vector pVL1393.
9.4. Analysis of Particle Production
[0380] AAV particles were produced as described in 1.1. Cell lysates were investigated by Western blot analysis for protein expression. pVL_VP1_MOD4 showed only VP3 expression, pVL_VP2 VP2 expression, while pVL_VP3 showed in addition to VP3 smaller degradation signals (
Conclusion
[0381] This result shows that AAV VLPs can be produced in insect cells as efficiently as in mammalian cells. The data show that in insect cells the N-terminal sequence of VP3 also seems to be required and sufficient for efficient VP3 capsid assembly. Further a change of the VP2 start codon from ACG into ATG comes along with loss of efficiency in capsid assembly (
10. Capsids Composed Essentially of VP3 Tolerate Insertions of Polypeptides
10.1. Generation of Virus-Like Particles (VLP) Containing Epitopes at Position I-587
[0382] For cloning of expression vectors encoding VLPs composed of VP3 capsid proteins containing a particular epitope sequence at position I-587, the epitope sequence was first cloned into the VP coding sequence of pUCAV2 at the site corresponding to I-587 (amino acid number relative to the VP1 protein of AAV-2) and was subsequently sub-cloned into the vector pCIVP2mutACG.
[0383] Generation of vector pUCAV2 is described in detail in U.S. Pat. No. 6,846,665. Basically, this vector contains the complete AAV2 genome (BgIII fragment) derived from pAV2 (Laughlin et al., 1983) cloned into the BamHI restriction site of pUC19. pUCAV2 was further modified by introduction of a NotI and AscI restriction site allowing the insertion of epitope sequences at position I-587 of the AAV2 capsid (PCT/EP2008/004366). In addition, an FseI restriction site located between position 453 (amino acid number relative to the VP1 protein of AAV-2) and I-587 was introduced in-frame into the VP coding sequence of the vector by site directed mutagenesis.
[0384] For cloning of epitope sequences into modified pUCAV2 sense- and anti-sense oligonucleotides were designed that encode the respective epitope with an alanine or glycine adaptor sequence and contain a 5′-site extension. The 5′-site extension of the oligonucleotides was designed so that annealing of the sense and anti-sense oligonucleotides results in a dsDNA with 5′-site and 3′-site overhangs compatible with overhangs generated by NotI and AscI restriction of the modified pUCAV2. The sequences of the oligonucleotides and the respective epitope sequences are summarized in Table 4. Each of the inserted epitopes is flanked by an adaptor according to the following scheme (X.sub.n represents the epitope sequence): [0385] Type I adaptor: (Ala).sub.2-(Gly).sub.3-X.sub.n-(Gly).sub.4-Ala [0386] Type II adaptor: (Ala).sub.2-(Gly).sub.4-X.sub.n-(Gly).sub.4-Ala [0387] Type III adaptor: (Ala).sub.3-(Gly).sub.5-X.sub.n-(Gly).sub.5-(Ala).sub.2 [0388] Type IV adaptor: (Ala).sub.5-X.sub.n-(Ala).sub.5
[0389] To anneal the oligonucleotides 50.0 μg of the sense oligonucleotide and 50.0 μg of the anti-sense oligonucleotide were mixed in a total volume of 200 μl 1×PCR-Buffer (Qiagen) and incubated for 3 min at 95° C. in a thermomixer. After 3 min at 95° C. the thermomixer was switched off and the tubes were left in the incubator for an additional 2 h to allow annealing of the oligonucleotides during the cooling down of the incubator. To clone the annealed oligonucleotides into pUCAV2 at I-587 the vector was linearized by restriction with NotI and AscI and the cloning reaction was performed using the Rapid DNA Ligation Kit (Roche). Briefly, the annealed oligonucleotides were diluted 10-fold in 1×DNA Dilution Buffer and incubated for 5 min at 50° C. 100 ng of the annealed oligonucleotides and 50 ng of the Not//AscI linearized vector pUCAV2 were used in the ligation reaction, which was performed according to the instructions of the manufacturer of the Rapid DNA Ligation Kit (Roche). E. coli XL1 blue or DH5a were transformed with an aliquot of the ligation reaction and plated on LB-Amp agar plates. Plasmids were prepared according to standard procedures and were analyzed by sequencing.
[0390] For generation of empty VLPs composed of VP3 proteins containing an epitope sequence at I-587 the BsiWI/XcmI restriction fragment of pUCAV2 containing the epitope at I-587 was sub-cloned into the vector pCIVP2mutACG according to standard procedures. The vector pCIVP2mutACG contains the overlapping AAV2 VP2 and VP3 coding sequences cloned into the XhoI/NotI site of pCI (Promega). In pCIVP2mutACG the ACG start-codon of VP2 is destroyed and replaced by a GAG codon. Substitution was performed by PCR amplification of the AAV2 VP2 and VP3 coding sequences using VP2 specific primers and the plasmid pCIVP2 as template (the vector pCIVP2 contains the wildtype VP2 and VP3 coding sequence cloned into the polylinker of pCI). The forward primer used for PCR anneals to the 5′ site of the VP2 coding sequence and contains the substitution of the VP2 ACG start codon by a GAG codon. In addition, the forward primer contains an XhoI recognition sequence at the 5′-site. The reverse primer annealed to the 3′ end of the VP2/VP3 coding sequence and contained a NotI recognition sequence at its 5′-site. The resulting PCR product was cloned into the XhoI/NotI site of pCI.
[0391] The resulting vectors were used for production of VLPs by transfection of 293-T cells. Cells (5×10.sup.5/dish) were seeded in 6 cm dishes 24 h prior to transfection. 293-T cells were transfected by calcium phosphate precipitation as described in US 2004/0053410. Subsequently, 293-T cells were lysed in the medium by three rounds of freeze (−80° C.) and thaw (37° C.) cycles. The lysate (3 ml total volume) was cleared by centrifugation and the VLP capsid titer was determined using a commercially available ELISA (AAV Titration ELISA; Progen, Heidelberg, Germany). VLP titers ranged between 2.1 E+12 and 9.8 E+12 capsids/ml (Table 5). The VLP TP18 clone was directly used for large scale packaging (as described in example 1). It contained 1.2E+13 capsids/ml within the crude lysate (Table 5).
10.2. Generation of Virus-Like Particles (VLP) Containing Epitopes at Position I-587 and I-453 of the Capsid
[0392] For cloning of expression vectors encoding VLPs composed of VP3 capsid proteins containing epitope sequences at position I-453 and I-587 (amino acid number relative to the VP1 protein of AAV-2), the first epitope sequence was cloned into pCIVP2mutACG at the site corresponding to I-587 as described above.
[0393] The second epitope sequence was initially cloned into the NotI/AscI restriction site of the vector pCIVP2-I453-NotI-AscI (described in: WO 2008/145400). Briefly, the vector pCI-VP2-I453-Not-AscI was created by PCR amplification of the AAV2 VP2 gene and cloning of the respective PCR product into the XhoI/NotI site of vector pCI (Promega). The resulting vector pCIVP2 was modified by destruction of the NotI restriction site of the cloning site by site-directed mutagenesis. The vector was further modified by introduction of a novel singular NotI and AscI restriction site allowing the insertion of epitope sequences at position I-453 of the AAV2 capsid. In addition, an FseI site located between I-453 and I-587 was introduced in-frame into the VP coding sequence of pCIVP2-I453-NotI-AscI by site directed mutagenesis. For cloning of epitope sequences into the NotI/AscI site of the vector sense- and anti-sense oligonucleotides were designed that encode the respective epitope with a alanine/glycine adaptor sequence and contain a 5′-site extension. The 5′-site extension of the oligonucleotides was designed so that annealing of the sense and anti-sense oligonucleotides results in a dsDNA with 5′-site and 3′-site overhangs compatible with overhangs generated by NotI and AscI restriction of pCIVP2-I453-Not-AscI. Cloning of the annealed oligonucleotides was performed as described above.
[0394] The sequences of the oligonucleotides and the respective epitope sequences are summarized in Table 6. Each of the inserted epitopes is flanked by an adaptor according to the following scheme (X.sub.n represents the epitope sequence): [0395] (Ala).sub.2-(Gly).sub.3-X.sub.n-(Gly).sub.4-Ala
[0396] For generation of bivalent VLPs displaying epitopes (murine TNFα or IL-17 epitope) at I-453 and I-587 the BsiWI/FseI fragment of pCIVP2-I453-NotI-AscI containing a given epitope inserted at I-453 was subcloned into the vector pCIVP2mutACG containing a particular epitope inserted into I-587 (described above). The resulting vector was used for production of bivalent VLPs by transfection of 293-T cells as described above (example 1.2) (6-well plate scale). Particle production was analyzed by ELISA (AAV2 Titration ELISA; Progen). Results are shown in Table 7. These data demonstrate that VLPs composed of VP3 proteins with epitope insertions at I-453 and I-587 can be produced with high capsid titers.
TABLE-US-00017 TABLE 4 Oligonucleotides used for cloning of epitope sequences into I-587 Name/ sense anti-sense Peptide Seq. Type Oligonucleotide Oligonucleotide Adaptor CETP TP18 Rabbit 5′GGCCGGCGGAGGTGACAT 5′CGCGCACCGCCACCCCC Type I DISVTGAPVIT CETP CAGCGTGACCGGTGCACCCG CAGGTAGGTGGCGGTGATC ATYL epitope TGATCACCGCCACCTACCTG ACGGGTGCACCGGTCACGC GGGGGTGGCGGTG 3′ TGATGTCACCTCCGCC 3′ (SEQ ID NO: 85) (SEQ ID NO: 86) 3Depi-3 Human 5′GGCCGGCGGAGGTGGTGA 5′CGCGCACCGCCACCCCC Type II DSNPRGVSAY IgE CAGCAACCCTAGAGGCGTGA TCTGCTCAGGTAGGCGCTC LSR epitope GCGCCTACCTGAGCAGAGGG ACGCCTCTAGGGTTGCTGT GGTGGCGGTG 3′ CACCACCTCCGCC 3′ (SEQ ID NO: 87) (SEQ ID NO: 88) Kricek Human 5′GGCCGCAGCGGCGGTGAA 5′CGCGCCGCCGCCGCCGC Type IV VNLTWSRASG IgE CCTGACCTGGAGCAGAGCCT GCCGGAGGCTCTGCTCCAG epitope CCGGCGCGGCGGCGGCGG GTCAGGTTCACCGCCGCTG 3′ (SEQ ID NO: 89) C3′ (SEQ ID NO: 90) TNFα-V1 Murine 5′GGCCGGCGGAGGTAGCAG 5′CGCGCACCGCCACCCCC Type I SSQNSSDKPV TNFα CCAGAACAGCAGCGACAAGC CTCCACCTGGTGGTTAGCC AHVVANHQVE epitope CCGTGGCCCACGTGGTGGCT ACCACGTGGGCCACGGGCT AACCACCAGGTGGAGGGGGG TGTCGCTGCTGTTCTGGCT TGGCGGTG 3′ GCTACCTCCGCC 3′ (SEQ ID NO: 91) (SEQ ID NO: 92) IL-17-V1 Murine 5′GGCCGGCGGAGGTAACGC 5′CGCGCACCGCCACCCCC Type I NAEGKLDHH IL-17 CGAGGGCAAGCTTGACCACC CAGCACGCTGTTCATGTGG MNSVL epitope ACATGAACAGCGTGCTGGGG TGGTCAAGCTTGCCCTCGG GGTGGCGGTG 3′ CGTTACCTCCGCC 3′ (SEQ ID NO: 93) (SEQ ID NO: 94) IL-6-V2 Murine 5′GGCCGGCGGAGGTCTGGA 5′CGCGCACCGCCACCCCC Type I LEEFLKVTLRS IL-6 GGAATTCCTGAAGGTGACCC GCTTCTCAGGGTCACCTTC epitope TGAGAAGCGGGGGTGGCGGT AGGAATTCCTCCAGACCTC G 3′ CGCC 3′ (SEQ ID NO: 95) (SEQ ID NO: 96) Aβ(1-9) Human 5′GGCCGCAGGCGGAGGGGG 5′CGCGCCGCGCCTCCCCC Type III DAEFRHDSG amyloid- AGGCGACGCCGAGTTCAGAC TCCGCCGCCGCTGTCGTGT β epitope ACGACAGCGGCGGCGGAGGG CTGAACTCGGCGTCGCCTC GGAGGCGCGG 3′ CCCCTCCGCCTGC 3′ (SEQ ID NO: 97) (SEQ ID NO: 98)
TABLE-US-00018 TABLE 5 Small scale production of different VLPs Titer (capsids/ Name Epitope at I-587 ml) VLP-TP18 CETP TP18 1.2E+13(*) DISVTGAPVITATYL (SEQ ID NO: 99) VLP- 3Depi-3 2.1E+12 3Depi3 DSNPRGVSAYLSR (SEQ ID NO: 100) VLP- Kricek 2.6E+12 Kricek VNLTWSRASG (SEQ ID NO: 101) VLP-TNFα TNFα-V1 9.8E+12 SSQNSSDKPVAHVVANHQVE (SEQ ID NO: 102) VLP-IL- IL-17-V1 5.6E+12 17 NAEGKLDHHMNSVL (SEQ ID NO: 103) VLP-IL-6 IL-6-V2 5.6E+12 LEEFLKVTLRS (SEQ ID NO: 104) VLP-Aβ Aβ(1-9) 6.2E+12 DAEFRHDSG (SEQ ID NO: 105) (*)Large-scale packaging
TABLE-US-00019 TABLE 6 Oligonucleotides used for cloning of epitope sequences into I-453 Name/ sense anti-sense Peptide Seq. Type Oligonucleotide Oligonucleotide TNFα-V1 Murine 5′GGCCGCCGGTGGAGGCAG 5′CGCGCCCTCCACCGCCCTCCAC SSQNSSDKPVA TNFα CAGCCAGAACAGCAGCGACA CTGGTGGTTAGCCACCACGTGGGC HVVANHQVE epitope AGCCCGTGGCCCACGTGGTG CACGGGCTTGTCGCTGCTGTTCTG GCTAACCACCAGGTGGAGGG GCTGCTGCCTCCACCGGC 3′ CGGTGGAGGG 3′ (SEQ ID NO: 107) (SEQ ID NO: 106) IL-17-V1 Murine 5′GGCCGCCGGTGGAGGCAA 5′CGCGCCCTCCACCGCCCAGCAC NAEGKLDHHMN IL-17 CGCCGAGGGCAAGCTTGACC GCTGTTCATGTGGTGGTCAAGCTT SVL epitope ACCACATGAACAGCGTGCTG GCCCTCGGCGTTGCCTCCACCGGC GGCGGTGGAGGG 3′ 3′ (SEQ ID NO: 108) (SEQ ID NO: 109) IL-6-V2 Murine 5′GGCCGCCGGTGGAGGCCT 5′CGCGCCCTCCACCGCCGCTTCT LEEFLKVTLRS IL-6 GGAGGAATTCCTGAAGGTGA CAGGGTCACCTTCAGGAATTCCTC epitope CCCTGAGAAGCGGCGGTGGA CAGGCCTCCACCGGC 3′ GGG 3′ (SEQ ID NO: 110) (SEQ ID NO: 111)
TABLE-US-00020 TABLE 7 Production of VLPs carrying epitopes at I-453 and I-587 Titer (capsids/ combination Epitope at I-453 Epitope at I-587 ml) TNF-α/IL-17 TNF α-V1 IL-17-V1 7.9E+12 SSQNSSDKPVAHVVANHQVE NAEGKLDHHMNSVL (SEQ ID NO: 112) (SEQ ID NO: 113) TNF-α/IL-6 TNF α-V1 IL-6-V2 8.5E+12 SSQNSSDKPVAHVVANHQVE LEEFLKVTLRS (SEQ ID NO: 114) (SEQ ID NO: 115) IL-17/TNF-α IL-17-V1 TNFα-V1 1.0E+13 NAEGKLDHHMNSVL SSQNSSDKPVAHVVANHQVE (SEQ ID NO: 116) (SEQ ID NO: 117) IL-6/TNF-α IL-6-V2 TNFα-V1 1.0E+13 LEEFLKVTLRS SSQNSSDKPVAHVVANHQVE (SEQ ID NO: 118) (SEQ ID NO: 119) IL-17/IL-6 IL-17-V1 IL-6-V2 3.9E+12 NAEGKLDHHMNSVL LEEFLKVTLRS (SEQ ID NO: 120) (SEQ ID NO: 121) IL-6/IL-17 IL-6-V2 IL-17-V1 8.9E+12 LEEFLKVTLRS NAEGKLDHHMNSVL (SEQ ID NO: 122) (SEQ ID NO: 123)
10.3. Conclusion
[0397] VP3 particles tolerate insertions and can therefore be used as a medicament such as a vaccine for example by insertion of B-Cell epitopes.
11. VP3 Capsid Assembly of Different AAV Serotypes
11.1. AAV1 Deletion Constructs
[0398] To analyze whether these findings can be conferred to other serotypes an analogue setting of constructs for AAV1 were tested.
[0399] Following constructs were cloned: [0400] pCI_VP2/2539_AAV1: The complete AAV1 VP2 plus 95 bp of VP1 were cloned into pCI (Promega, Mannheim, Germany). The VP2 ACG start codon was not mutated. [0401] pCI_VP3/2539_AAV1 mutACG: The complete AAV1 VP2 plus 95 bp of VP1 were cloned into pCI. The VP2 ACG start codon was mutated to ACC. [0402] pCI_VP3/2634_AAV1 mutACG: The VP1 part was deleted completely and the VP2 ACG start codon was mutated into an ACC.
Cloning
[0403] Cloning of all constructs was performed by site directed mutagenesis standard procedures using modified primers (primers used for site directed mutagenesis are listed below). pCI_VP2/2539_AAV1 was generated by inserting a NheI site 95 bp upstream of the VP2 ACG start codon and a XmaI site downstream of the VP3 stop codon. Mutations were generated within pUCrep/fs/cap_AAV1_1588 (described within PCT/EP2008/004366). The resulting plasmid was digested with NheI and XmaI. The generated fragment was cloned into the pCI-VP2 Vector (described in PCT/EP2008/004366). Primers:
TABLE-US-00021 AAV1 NheI VP2plus95bp: (SEQ ID NO: 124) 5′-GAG CGT CTG CTA GCA GAT ACC TCT TTT GGG G-3′ AAV1 VP3 Xma rev: (SEQ ID NO: 125) 5′-GAA ACG AAT CAC CCG GGT TAT TGA TTA AC-3′
pCI_VP3/2539_AAV1 mutACG was generated by mutating the ACG start codon to ACC within pCIVP2/2539_AAV1. Primer:
TABLE-US-00022 AAV1 VP2ko for: (SEQ ID NO: 126) 5′-GGC GCT AAG ACC GCT CCT GGA AAG-3′ AAV1 VP2ko rev: (SEQ ID NO: 127) 5′-CTT TCC AGG AGC GGT CTT AGC GCC-3′
pCI_VP3/2634_AAV1 mutACG was generated by deleting the 95 bp directly upstream of the VP2 ACG start codon and mutating by the same step the ACG start codon to ACC within pCIVP2_AAV1. Primer:
TABLE-US-00023 AAV1 VP2ko_VP1del for: (SEQ ID NO: 128) 5′-ACG ACT CAC TAT AGG CTA GCA GGC GCT AAG ACC GCT CCT GGA AAG-3′ AAV1 VP2ko_VP1del rev: (SEQ ID NO: 129) 5′-CTT TCC AGG AGC GGT CTT AGC GCC TGC TAG CCT ATA GTG AGT CGT-3′
[0404] Assembly of AAV1 capsids was controlled within crude lysates after transfection of 293 cells with the respective plasmid. The capsid titer was determined by an AAV1 titration ELISA (Progen, Heidelberg, Germany) according to manufacturer's manual. The assembly efficiency of the three AAV1 constructs was comparable. The construct pCI_VP3/2634_AAV1 mutACG gave a titer of 10.sup.13 particles/ml, confirming the fact that capsid generation of AAV1 particles is generally more efficient than of AAV2 particles. In Western blot analyses VP2 and VP3 proteins were detectable for construct pCI_VP2/2539_AAV1 and only VP3 was detectable for pCI_VP3/2539_AAV1 mutACG and pCI_VP3/2634_AAV1 mutACG respectively (
[0405] As a control for capsid protein expression, pUCAV1 was transfected. pUCAV1 contains the complete AAV1 Cap open reading frame encoding VP1, VP2 and VP3 of AAV1. pUCAV1 is described in detail in the PCT submission PCT/EP2008/004366 (there referred to as “pUCAV1_AgeI”).
11.2. Trans-Complementation of pCMV Driven AAV1 VP3 Constructs
[0406] To see whether trans-complementation experiments described in example 5 can be conferred to other serotypes analogue constructs of pCMV-VP3/2809 (AAV2) were cloned for AAV1.
11.2.1. Cloning
[0407] pCMV_AAV1VP3/2829 was cloned as following: By mutagenesis a HindIII restriction site was introduced directly before the VP3 ATG start codon of plasmid pUCrep/fs/cap_AAV1 (described within PCT/EP2008/004366) using the primers indicated below. The resulting plasmid was digested with AgeI. The Age I site was blunt ended with Klenow polymerase and the construct was subsequently digested with HindIII. The generated fragment was cloned into the HindIII/HincII-digested pBSCMV backbone. pBSCMV was generated by insertion of a 650 bp BamHI CMV promoter fragment into the BamHI site of BlueskriptII SK+ vector (Stratagene, Amsterdam, Netherlands) described by Wistuba et al, 1997. Primer Hind III mutagenesis:
TABLE-US-00024 Forward: (SEQ ID NO: 130) 5′-CGC TGC TGT GGG ACC TAA GCT TAT GGC TTC AGG CGG TGG CG-3′ Reverse: (SEQ ID NO: 131) 5′-CGC CAC CGC CTG AAG CCA TAA GCT TAG GTC CCA CAG CAG CG-3′
11.2.2. Trans-Complementation Assay
[0408] Trans-complementation was performed with the pVP2N-gfp construct from AAV2 as described in example 3. Cells were transfected with plasmid pCMV-VP3 of either AAV2 pCMV_VP3/2809) or AAV1 (pCMV_AAV1VP3/2829) with or without cotransfection of pVP2N-gfp (
11.2.3. Result and Conclusion
[0409] Particle assembly of AAV1 analyzed by an AAV1 ELISA (Progen, Heidelberg) was rescued by trans-complementation with pVP2N-gfp derived from AAV2. Rescue efficiency cannot be indicated as we did not compare cotransfection of pCMV_AAV1VP3/2829 and pVP2N-gfp with transfection of pCIVP3/2634_AAV1 mutACG (see chapter 11.1 above). Also, we did not yet clone and test an AAV1 trans-complementation plasmid pVP2N-Gfp
[0410] Particle titer measured for trans-complemented AAV2 VP3 was 2.1E11. For AAV1 VP3 the titer obtained was 3.4E10 (a direct comparison of AAV1 and AAV2 titers is not possible due to the use of different ELISAs).
[0411] The results indicate that AAV1 makes use of the same mechanism for capsid assembly as AAV2 and that fragment Z and VP3 are interchangeable with different AAV serotypes.
11.3. Insertion of Polypeptides within AAV1 I588 is Tolerated
[0412] Here it was investigated whether empty AAV1 essentially VP3 particles tolerate insertions within amino-acid position 588.
[0413] For cloning of epitope sequences into pUCAV1-AgeI-1588 (described in PCT/EP2008/004366), sense- and anti-sense oligonucleotides were designed that encode the respective epitope with a glycine adaptor sequence. Upon hybridization of both oligonucleotides, 5′- and 3′-overhangs are generated that are compatible with overhangs generated by NotI and AscI restriction of the pUCAV1-AgeI-1588. The sequences of the oligonucleotides and the respective epitope sequences investigated are summarized in Table 4. Each of the inserted epitopes is flanked by an adaptor according to the following scheme (X.sub.n represents the epitope sequence): Ser(588)-(Ala).sub.2-(Gly).sub.5-X.sub.n-(Gly).sub.5-Thr(589)
[0414] Oligo nucleotides for cloning the human IgE epitope “Kricek”
TABLE-US-00025 Amino acid sequence: VNLTWSRASG Sense oligo: (SEQ ID NO: 132) 5′-g gcc gca gcc gca gtg aac ctg acc tgg agc aga gcc tcc ggc gcg gca gct gca gct-3′ antisense oligo: (SEQ ID NO: 133) 5′-g gcg agc tgc agc tgc cgc gcc gga ggc tct gct cca ggt cag gtt cac tgc ggc tgc-3′
[0415] Oligo nucleotides for cloning the human IgE epitope “3Depi-3”
TABLE-US-00026 Amino acid sequence: DSNPRGVSAYLSR Sense oligo: (SEQ ID NO: 134) 5′-GGCC GGC GGT GGA GGC GGT GAC AGC AAC CCT AGA GGC GTG AGC GCC TAC CTG AGC AGA GGA GGC GGT GGA GGG-3′ antisense oligo: (SEQ ID NO: 135) 5′-CGCG CCC TCC ACC GCC TCC TCT GCT CAG GTA GGC GCT CAC GCC TCT AGG GTT GCT GTC ACC GCC TCC ACC GCC-3′
[0416] The precise cloning procedure used corresponds to the protocol used for insertion of epitopes into AAV2 I587 described in example 10.
[0417] For generation of empty AAV1 VLPs composed of essentially VP3 proteins containing an epitope sequence at I-588 the BsiWI/SphI restriction fragment of pUCAV1-AgeI-1588 carrying the epitope at I-588 was sub-cloned into the vector pCIVP3/2634_AAV1 mutACG (described in example 11.1) according to standard procedures.
[0418] The resulting vectors were used for production of AAV1 VLPs by transfection of 293-T cells as described above (example 1.2.)
[0419] Titers were determined by a commercial AAV1 ELISA (Progen, Heidelberg, Germany). High titers of 3.6E13/ml (Kricek) and 9.2E13/ml (3Depi-3) were obtained, indicating that insertions within AAV1 588 (being homologous to AAV2 587) are well tolerated and that AAV1 VP3 particles can be used as vaccine carrier.
12. ORF2 Comprises Fragment Z and Encodes AAP.
[0420] Detailed sequence analysis revealed that fragment Z encodes a significant part of the new “assembly activating protein” (AAP).
[0421] In
[0422] The sequences of the respective open reading frames and proteins of some other parvoviruses were extracted from the capsid gene sequences available in the NCBI database and given in detail in SEQ ID Nos 2-44 as listed in table 8.
TABLE-US-00027 TABLE 8 NCBI entrée numbers and numbers of corresponding SEQ IDs of AAP encoding nucleotide and protein sequences from different parvoviruses. No. of nt respective Length of encoded protein Length of parvovirus entrée at NCBI ORF2 ORF2/nt AAP AAP/AA AAV2 NC_001401 SEQ ID NO: 23 627 SEQ ID NO: 1 208 AAV1 NC_002077 SEQ ID NO: 24 678 SEQ ID NO: 2 225 AAV3b AF028705 SEQ ID NO: 25 627 SEQ ID NO: 3 208 AAV4 NC_001829 SEQ ID NO: 26 597 SEQ ID NO: 4 198 AAV5 NC_006152 SEQ ID NO: 27 681 SEQ ID NO: 5 226 AAV6 AF028704 SEQ ID NO: 28 678 SEQ ID NO: 6 225 AAV7 NC_006260 SEQ ID NO: 29 681 SEQ ID NO: 7 226 AAV8 NC_006261 SEQ ID NO: 30 684 SEQ ID NO: 8 227 AAV9 AY530579 SEQ ID NO: 31 681 SEQ ID NO: 9 226 AAV10 AY631965 SEQ ID NO: 32 606 SEQ ID NO: 10 201 AAV11 AY631966 SEQ ID NO: 33 594 SEQ ID NO: 11 197 AAV12 DQ813647 SEQ ID NO: 34 621 SEQ ID NO: 12 206 b-AAV (bovine) NC_005889 SEQ ID NO: 35 600 SEQ ID NO: 13 199 Avian AAV AY186198 SEQ ID NO: 36 789 SEQ ID NO: 14 262 ATCC VR-865 Avian AAV AY629583 SEQ ID NO: 142 723 SEQ ID NO: 143 240 strain DA-1 AAV13 EU285562 SEQ ID NO: 37 627 SEQ ID NO: 15 208 Mouse AAV1 DQ100362 SEQ ID NO: 38 534 SEQ ID NO: 16 177 Avian AAV AY629583 SEQ ID NO: 39 723 SEQ ID NO: 17 240 strain DA-1 Caprine AAV1 AY724675 SEQ ID NO: 40 581 SEQ ID NO: 18 226 isolate AAV-Go. 1 Rat AAV1 DQ100363 SEQ ID NO: 41 756 SEQ ID NO: 19 251 Goose EU088102 SEQ ID NO: 42 639 SEQ ID NO: 20 212 parvovirus strain DB3 Duck AY382892 SEQ ID NO: 43 693 SEQ ID NO: 21 230 parvovirus strain 90-0219 Snake AY349010 SEQ ID NO: 44 600 SEQ ID NO: 22 199 parvovirus 1
[0423] For sequence comparison an alignment of the predicted AAP protein sequences derived from ORF2 of the cap gene of some parvoviruses is given in
[0424] In construct pVP2N-gfp the EcoNI/BsiWI fragment from pTAV2.0 was inserted downstream of a CMV promoter and upstream of the GFP cds of vector pEGFP-N1 (example 3.1/
13. Codon Modification Confirms that Expression of Functional Protein from ORF2 is Necessary for Trans-Complementation
[0425] To investigate the nature of the trans-complementing activity of ORF2, the sequence between the EcoNI/BsiWI restriction fragment was codon modified (cm).
[0426] The first mutant DNA sequence was named ORF1 cm. The DNA sequence of the mutant was altered in such a way that the first reading frame coding for the capsid protein remained intact whereas the second reading frame coding for AAP was changed. As a result the sequence encodes wildtype capsid protein but no functionally active AAP any more. Identity of the DNA sequence of pVP2N-gfp versus pVP2N/ORF1cm-gfp is 71% while protein identity in the first reading frame is 100%.
[0427] The second mutant DNA sequence was named ORF2 cm and altered in the first reading frame meaning that it did not code for a functionally active capsid protein any more but functionally intact AAP could be expressed. Identity of the DNA sequence of pVP2N-gfp versus pVP2N/ORF2 cm-gfp is 79% while protein identity in the second reading frame is 100%.
[0428] The sequences of ORF1cm and ORF2 cm are given in
[0429] As described in example 3.1, pVP2N-gfp was generated by inserting the EcoNI/BsiWI restriction fragment of pTAV2.0 into the multiple cloning site of pEGFP-N1. Constructs pVP2N/ORF1cm-gfp and pVP2N/ORF2 cm-gfp were generated in the same way with the difference that the codon modified EcoNI/BsiWI fragments were inserted into the corresponding vector backbone.
[0430] Protein expression of pVP2N/ORF1cm-gfp and pVP2N/ORF2 cm-gfp (
Result and Conclusion
[0431] As already described in example 3 and shown in
[0432] Hence it was clear, that the assembly promoting activity associated with the constructs containing cap sequences upstream of the VP3 translation start site can be provided in trans.
[0433] As already described for example 5,
[0434] In contrast, assembled capsid could be detected using the helper construct pVP2N/ORF2 cm (
[0435] This result clearly indicates, that the trans-complementing activity of fragment Z is mediated by its encoded protein AAP in ORF2. Codon modification experiments confirmed that expression of functional capsid protein in ORF1 is not necessary for trans-complementation but expression of functional AAP in ORF2.
14. Mutation of the Predicted Translation Start Codon of AAP
[0436] The sequence of ORF2 as given in
[0437] Protein expression of AU1 tagged versions of ORF2, namely pORF2/CTG-AU1, pORF2/ATG-AU1 and pORF2/TTG-AU1 (
[0438] Constructs pORF2/CTG-AU1, pORF2/ATG-AU1 and pORF2/TTG-AU1 comprise the entire ORF2 of the cap gene (AAV2 nt 2717-3340) fused to sequences coding for an AU1-tag (FIG. A).
[0439] For generation of constructs pORF2/CTG-AU1, pORF2/ATG-AU1 and pORF2/TTG-AU1 PCRs were performed with template pTAV2.0 and forward primer
TABLE-US-00028 (SEQ ID NO: 136) 5′-GGATCGCAAGCTTATTTTGGTCAGACTGGAGACGCAGACTCAGTACC TGACCC-3′, (SEQ ID NO: 137) 5′-GGATCGCAAGCTTATTTTGGTCAGAATGGAGACGCAGACTCAG-3′,
or
TABLE-US-00029 (SEQ ID NO: 138) 5′-GGATCGCAAGCTTATTTTGGTCAGATTGGAGACGCAGACTCAG-3′
and reverse primer
TABLE-US-00030 (SEQ ID NO: 139) 5′-GCGGTGTCTCGAGTTATATATAGCGATAGGTGTCGGGTGAGGTATCC ATACTGTGGCACCATGAAGAC-3′.
[0440] The HindIII/XhoI digested amplification products were inserted into the HindIII/XhoI backbone of pBS-CMVsense, which was generated by insertion of a 560 bp BamHI human cytomegalovirus (CMV) promoter fragment from pHCMV-Luci (kindly provided by K. Butz, Germen Cancer Research Center, Heidelberg, Germany) into the BamHI site of plasmid Bluescript II SK+(pBS, Stratagene, La Jolla, Calif., USA).
Results and Conclusion
[0441] The expression of the postulated proteins could be demonstrated using a monoclonal antibody against the AU1-tag (anti-AU1) for the constructs pORF2/CTG-AU1 and pORF2/ATG-AU1 (
[0442] Taken together, mutation of the putative non-canonical CTG start codon into a strong ATG start codon enhanced protein synthesis and capsid assembly whereas mutation into a codon which normally is not preferred as initiation codon for protein synthesis significantly reduces protein levels and the number of assembled capsids. This result not only corroborates our conclusion that the protein product of ORF2 promotes the capsid assembly process. The results further indicate that the non-canonical CTG start codon is likely used as a start for translation, as its mutation into TTG leads to a significant reduction of AAP expression.
15. Insertion of Stop Codons in ORF2 Confirm that Expression of Functional AAP is Necessary for Trans-Complementation
[0443] Additionally, mutations were performed in the AAP encoding reading frame by introduction of stop codons into ORF2 in order to confirm that expression of functional AAP is necessary for trans-complementation.
[0444] Plasmids pVP2N/ORF2stopA-gfp, pVP2N/ORF2stopB-gfp, and pVP2N/ORF2stopC-gfp were created by site-directed mutagenesis (QuickChange site-directed mutagenesis kit, Stratagene) of template pVP2N-gfp using two complementary PCR primers which included the desired substitutions. In pVP2N/ORF2stopA-gfp codon tgg.sub.2811 has been mutated into tag, in pVP2N/ORF2stopB-gfp codon c.sub.2831aa has been mutated into taa, and in pVP2N/ORF2stopC-gfp codon g.sub.2879aa has been mutated into tga (
Results and Conclusion
[0445] Western blot analysis confirmed that VP3 is expressed in all samples (detected by monoclonal antibody B1 in
[0446] Accordingly, Cap expression from pVP2n-gfp is not sufficient for capsid assembly in the trans-complementation assay. This result clearly supports the existence of AAP expressed from a different reading frame (ORF2) overlapping with the cap gene, which provides the capsid assembly helper function.
16. Expression of Functional AAP Rescues Capsid Assembly in the Context of the AAV Genome
[0447] Next we wanted to analyze whether expression of the newly discovered “assembly activating protein” AAP is necessary for capsid assembly in the context of the whole AAV genome. Therefore, construct pTAV/ORF1cm was created by cloning the EcoNI/BsiWI fragment of pVP2N/ORF1cm-gfp (example 13) into the EcoNI/BsiWI backbone of pTAV2.0 (example 1.2.1.). Hence, plasmid pTAV/ORF1cm (schematically shown in
Results and Conclusion
[0448] Indeed, the four Rep proteins (Rep40, Rep52, Rep68, and Rep78) were correctly expressed (data not shown). Western blot analysis showed that the expression pattern of the three VP proteins was slightly altered. Expression of endogenous AAP from wildtype plasmid pTAV2.0 but not from the codon modified one pTAV/ORF1cm was directly proven using polyclonal anti-AAP serum (
[0449] Capsid assembly of the two constructs was compared after co-transfection of wildtype plasmid pTAV2.0 and codon modified plasmid pTAV/ORF1cm with empty Bluescript vector (pBS) or with pVP2N-gfp. As expected, transfection of pTAV/ORF1cm with pBS showed no detectable capsid formation, since pTAV/ORF1cm expresses all three capsid proteins but neither pTAV/ORF1 cm nor pBS express functionally active AAP. In contrast, transfection of pTAV/ORF1 cm with pVP2N-gfp restored capsid assembly at least partially (
[0450] Complementation of pTAV/ORF1cm that is deficient in expression of functional active AAP with mutant plasmids like pVP2N/ORF1cm-gfp (as described in example 13) and pVP2N/ORF2stopA-gfp (see example 15) which both were unable to express the AAP protein (due to codon modification or introduction of a stop codon, respectively) also did not lead to capsid formation. In contrast, in addition to pVP2N-gfp functionally active AAP can be expressed from plasmids pVP2N/ORF2 cm-gfp (described in example 13), pORF2/CTG-AU1 and pORF2/ATG-AU1 (see example 14) and rescued capsid assembly in trans-complementation (
[0451] Taken together, capsid formation in the context of the complete viral genome is dependent on the expression of endogenous or complemented AAP.
17. Expression of Functional AAP is Necessary for Capsid Assembly in the Context of the AAV Genome
[0452] To further prove that AAP is necessary for capsid assembly in the context of the whole AAV genome, a stop codon was introduced in ORF2 disrupting AAP amino acid sequence.
[0453] Therefore, construct pTAV/ORF2stopB was created by cloning the EcoNI/BsiWI fragment of pVP2N/ORF2stopB-gfp (for details see example 15) into the EcoNI/BsiWI backbone of pTAV2.0. (example 1.2.1). In pVP2N/ORF2stopB-gfp the caa codon starting at nucleotide was mutated into a taa stop codon. Hence, plasmid pTAV/ORF2stopB (schematically shown in
Results and Conclusion
[0454] Again, correct expression of the four Rep proteins could be detected in Western blot analysis (data not shown), as well as a slightly altered expression pattern of the three VP proteins. Expression of endogenous AAP from wildtype plasmid pTAV2.0 but not from the one containing the stop codon was directly proven using polyclonal anti-AAP serum (
[0455] Capsid assembly of the two constructs was compared after co-transfection of wildtype plasmid pTAV2.0 and mutant plasmid pTAV/ORF2stopB with empty Bluescript vector (pBS) or with pVP2N-gfp. As expected, transfection of pTAV/ORF2stopB with pBS showed no detectable capsid formation, since pTAV/ORF2stopB expresses all three capsid proteins but neither pTAV/ORF2stopB nor pBS express functionally active AAP. In contrast, transfection of pTAV/ORF2stopB with pVP2N-gfp restored capsid assembly at least partially (
[0456] This result further confirmed that capsid formation in the context of the complete viral genome is dependent on the expression of functional AAP.
18. The “Assembly Activating Protein” AAP Targets VP Proteins to the Nucleolus.
[0457] In addition to example 8, several constructs were transfected in 293-T cells to compare the location of expressed proteins within the transfected cell and assembly efficiency.
18.1. Cloning of Constructs
[0458] Cloning of construct pCMV-NLS-VP3 is described in example 8.1. The approach for generation of pCMV-NoLS-VP3 was concordant to that of pCMV-NLS-VP3 with the difference that the complementary primer pair
TABLE-US-00031 (SEQ ID NO: 140) 5′-GGAAT TCGAT ATCAA GCTTG CCATG GCACG GCAGG CCCGG CGGAA TAGAC GGAGA CGGTG GCGGG AACGG CAGCG GATGG CTACA GGCAG TGG-3′, and (SEQ ID NO: 141) 5′-CCACT GCCTG TAGCC ATCCG CTGCC GTTCC CGCCA CCGTC TCCGT CTATT CCGCC GGGCC TGCCG TGCCA TGGCA AGCTT GATAT CGAAT TCC-3′
was used. Accordingly, the cap gene product NoLS-VP3 contains the amino acid sequence of the nucleolar localization signal of HIV Rev MARQARRNRRRRWRERQR at the N terminus of VP3. Both constructs are schematically shown in
18.2. Analyses of Constructs by Immunofluorescence
[0459] Analogous to the experimental setup described in example 8, HeLa cells were transfected with the different constructs as indicated. Expression of capsid proteins and formation of capsids was analyzed by immunofluorescence as described above using a polyclonal VP antiserum or the monoclonal A20 antibody.
18.3. Results and Conclusion
[0460] From literature analyzing productive AAV infection (e.g. Wistuba et al., 1997) it is known that capsid assembly can first be detected in the nucleoli of infected cells. Capsid protein VP3 expressed from pCMV-VP3/2809 in HeLa cells was distributed throughout the cell nucleus and the cytoplasm and excluded from nucleoli (as shown in
[0461] As described in example 8, we expressed the construct pCMV-NLS-VP3 and observed strong nuclear accumulation of VP3 fused to the nuclear localization signal (NLS) of SV40, which however was excluded from nucleoli and did not cause capsid assembly (
[0462] Interestingly, AAP protein expressed from pORF2/ATG-AU1 (described in example 14) and stained with anti-AU1 antibody co-located with Fibrillarin to the nucleoli (
[0463] This result suggested that AAP co-transports VP proteins to the nucleoli, which is a prerequisite for subsequent capsid assembly.
[0464] When expressing the construct pCMV-NoLS-VP3 we observed at least partially nucleolar localization of VP3 fused to the nucleolar localization signal derived from HIV REV, but surprisingly no capsid assembly could be detected (
19. Expression of Functional AAP is Necessary for Capsid Assembly.
[0465] In addition to the immunofluorescence images seen in example 18 we analyzed protein expression of the respective mutant constructs pCMV-NLS-VP3 and pCMV-NoLS-VP3 on Western blots. Moreover, we quantified capsid assembly activity of the respective constructs by monoclonal antibody A20 capsid ELISA.
Results and Conclusion
[0466] Western blot analysis confirmed expression of VP3 from pCMV-VP3/2809 and the slightly longer proteins NLS-VP3 and NoLS-VP3 from pCMV-NLS-VP3 and pCMV-NoLS-VP3, respectively (
[0467] As already observed in example 18, neither NLS-VP3 nor NoLS-VP3 rescue capsid formation upon cotransfection with Bluescript vector (pBS), whereas in the presence of AAP expression (from pVP2N-gfp) capsid formation was detectable (
[0468] This result confirms that AAP not only targets VP proteins to the nucleoli (which is also accomplished by the NoLS-VP3 fusion construct not leading to capsid assembly) but also plays an essential role in the assembly reaction itself.
20. Assembly of Wildtype and VP3 VLPs
[0469] To compare the morphology of virus-like particles assembled of VP1, VP2 and VP3 (VP1,2,3 VLP) with that of VLPs assembled only of VP3 (VP3 VLP) the respective samples have been investigated by electron microscopy after negative staining using 2% uranylacetate as described above.
[0470] Virus-like particles assembled of VP1, VP2 and VP3 corresponding to the wildtype capsid were produced in 293-T cells by expression of the complete cap gene. VLPs assembled only of VP3 were produced by co-transfection of pCMV-VP3/2809 and pVP2N-gfp (VP3 VLP).
Results and Conclusion
[0471] Electron microscopic images confirmed that the morphology of virus-like particles assembled of VP1, VP2 and VP3 (VP1,2,3 VLP) is comparable to that of VLPs assembled only of VP3 (VP3 VLP,
21. Trans-Complementation of AAP and VP3 Cloned from Different Serotypes
[0472] To confirm that expression of AAP from one parvovirus is capable of mediating capsid assembly of VP3 from another parvovirus, we used the respective sequences of AAV1, AAV2 and AAV5 in trans-complementation assays.
[0473] Cloning of pVP2N-gfp of AAV1 and AAV5 was performed analogous to that of AAV2 (compare 3.1) with the difference that primer pairs were selected to amplify the respective sequences for AAV1 and AAV5 as given in SEQ ID NO: 24 and SEQ ID NO: 27 respectively. For trans-complementation cells were transfected with plasmid pCMV-VP3 of either AAV2 (pCMV_VP3/2809), AAV1 (pCMV_AAV1VP3/2829) as described above or a corresponding AAV5 VP3 expression construct with or without cotransfection of pVP2N-gfp of the respective AAV serotype (
Results and Conclusion
[0474] Capsid assembly of VP3 cloned from AAV1, AAV2 and AAV5, respectively, was compared after co-transfection of pVP2N-gfp cloned from AAV2 and AAV1, respectively, or Bluescript vector (pBS) (see
[0475] Still, in addition to example 11 these result confirm that parvoviruses other than AAV2 encode functional AAP and make use of the same mechanism for capsid assembly. Further, AAP and VP3 are in principal interchangeable between different parvoviruses, especially between closely related viruses.
LITERATURE
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