EP1084236A1 - Nouveaux vecteurs adenoviraux utilises en therapie genique - Google Patents

Nouveaux vecteurs adenoviraux utilises en therapie genique

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Publication number
EP1084236A1
EP1084236A1 EP99927250A EP99927250A EP1084236A1 EP 1084236 A1 EP1084236 A1 EP 1084236A1 EP 99927250 A EP99927250 A EP 99927250A EP 99927250 A EP99927250 A EP 99927250A EP 1084236 A1 EP1084236 A1 EP 1084236A1
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European Patent Office
Prior art keywords
vector
leptin
dna
expression cassette
heterologous expression
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EP99927250A
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German (de)
English (en)
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EP1084236A4 (fr
Inventor
Manal A. Morsy
Volker Sandig
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Merck and Co Inc
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Merck and Co Inc
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Publication of EP1084236A1 publication Critical patent/EP1084236A1/fr
Publication of EP1084236A4 publication Critical patent/EP1084236A4/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/2264—Obesity-gene products, e.g. leptin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/5759—Products of obesity genes, e.g. leptin, obese (OB), tub, fat
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/38—Vector systems having a special element relevant for transcription being a stuffer

Definitions

  • This invention related to adenoviral vectors which are useful as vectors for gene delivery in gene therapy applications.
  • Ad vectors are currently among the most efficient gene transfer vehicles for both in vitro and in vivo delivery, but the utilization of current Ad vectors for many gene therapy applications is limited by the transient nature of transgene expression obtained by these vectors.
  • Ad vectors that are deleted in all viral protein-coding sequences offers the prospect of a potentially safer, less immunogenic vector with an insert capacity of up to approximately 38 kb.
  • These vectors require supplementation by viral regulatory and structural proteins supplied in trans for packaging and rescue, and are thus termed "helper-dependent" (HD).
  • HD helper-dependent
  • both ends contained 5' regions, with two 3' regions joined in the center); and a "tail-to-tail" configuration (3'-5'-5'-3', i.e. both ends contained 3' regions, with two 5' regions joined in the center).
  • These viruses were present as a mixture of multimers of a small genome, wherein the only adenoviral sequences were the ITR and packaging regions. Parks et al, 1997, J Virology 71(4):3293-3298 describes a population of HD-adeno viruses in which a 15.1 kb virus concatamerized spontaneously in culture, and formed two different species of 30.2 kb dimers.
  • HD-adenoviruses as vectors for gene therapy is contamination of the HD-virus population with the helper viruses needed for viral culture and replication. It would be desirable to have a HD-system wherein helper virus contamination was low.
  • Figure 1 is a diagram of vectors HD-leptin-monomer and HD-2 ⁇ - leptin.
  • Figure 1 A is the DNA composite fragments of ⁇ STK120-HCMV-mOb- BGHpA (approximately 19.6 kb total size).
  • the fragments are: the left end terminus of Ad5, containing the inverted terminal repeat sequences and the packaging signal ⁇ (nucleotides (nt.) 1-440, solid arrow); the 5072 bp fragment of hypoxanthine guanine phosphoribosyltransferase (HPRT) (nt.12373-17853 in Genbank gb:humhprtb; shown as a bar) (inserted in the complementary orientation); the Hindlll 9063 bp fragment of C346 cosmid (nt.12421-21484 in gb:L31948, shaded bar); and the right end terminus of Ad5, composed of the ITR sequence (nt. 35818-35935).
  • the ITRs are flanked by unique Pmel restriction sites used to liberate the vector fragment from the plas id backbone prior to the initial transfection into 293-cre4 cells for viral rescue and propagation (released fragment is 16.7 kb).
  • Figure IB shows a virus according to this invention, HD-2 ⁇ -leptin, which is a tail-to-tail concatamerization of the virus of Figure 1 A.
  • Figure 2 is a series of gels demonstrating sizes of vectors made in accordance with this invention, and sizes of vectors cut with various restriction enzymes. Further details are given in the Examples.
  • Figure 3 is a diagram of vectors (A) stkl 20-1 ⁇ -CMV-hVEGF 145.
  • SV40pA and (B) stkl20-2 ⁇ -CMV-hVEGFl45-SV40pA From left to right the fragments in figure 3(A) are: the left end terminus of Ad5, containing the inverted terminal repeat sequences and the packaging signal ⁇ (nucleotides (nt.) 1-440, solid arrow); the 16054 bp fragment of hypoxanthine guanine phosphoribosyltransferase (HPRT) (nt.12373-17853 in Genbank gb:humhprtb; shown as a bar) (inserted in the complementary orientation); the Hindlll 9063 bp fragment of C346 cosmid (nt.12421- 21484 in gb:L31948, shaded bar); and the right end terminus of Ad5, composed of the ITR sequence (nt.
  • the fragments in (B) are identical to those in (A) exept for the right end terminus of Ad5 which is replaced by an inverted left end terminus, containing the inverted terminal repeat sequences and the packaging signal ⁇ (nucleotides (nt.) 1-440, solid arrow).
  • the ITRs are flanked by unique Pmel restriction sites used to liberate the vector fragment from the plasmid backbone prior to the initial transfection into 293-cre4 cells for viral rescue and propagation (released fragment is 29 kb).
  • Figure 4 shows a comparison of the vectors HD-l ⁇ and HD-2 ⁇ after propagation.
  • Stuffer DNA non-expressed DNA which is inserted to an adenoviral vector to increase the size of virus to at least about 75% of wild type, more preferably from at least about 75-105% wild-type (about 26 -38 kb).
  • HD-virus A "helper-dependent" virus: an adenovirus which has native genes deleted so that it cannot replicate unless the products of the deleted genes are provided by another source, such as a helper virus or by a cell line.
  • Expression Cassette contains a gene whose product is to be expressed, along with any and all necessary control sequences.
  • Tail-to-tail Concamerization two virus are joined so that their regions are 5'-3'-3*-5'.
  • Head-to-tail Concatamerization two viruses are joined so that their regions are 5'-3'-5'-3'.
  • Plasmid-based sequences refers to DNA and genes present in plasmids which are used during construction of the adenoviral vector, but which are not adenoviral DNA, nor the heterologous gene which is to be delivered by the adenoviral vector. Examples of plasmid-based sequences include: the origin of replication (o ), antibiotic resitance genes used as markers (ampicillin resistance), and the like. Adenoviral vectors are generally useful for delivering genes in gene therapy.
  • adenoviral vectors have been made which contain a deletion in the section of their genomes coding for replication-related functions, such as the El and E3 genes. These deletions have two different functions: aside from making a safe, non-replicating viral vector, the deletions allow room for insertion of transgenes.
  • Such viruses with deleted replication functions are generally referred to as helper-dependent (HD)-viruses. They cannot replicate on their own, but the replication-related functions can be provided for in trans by helper viruses which are co-cultivated with the HD- viruses. While propagating HD-viruses using conventional cell lines and helper viruses containing genes for viral replication, a spontaneous concatamerization occurred. These new HD-2 ⁇ -viruses were rescued and propagated, and were analyzed both for structure verification and for helper-load contamination. Althernatively, these viruses may be made by ligating together the various described segments, using conventional genetic engineering techniques.
  • the concatamerized HD-adenoviral vectors according to this invention contain the following elements:
  • the vector may contain a second heterologous expression cassette (F) between the first heterologous expression cassette (element C) and the second packaging signal (element D).
  • the first and second expression cassette may express the same gene, or they may express different gene products.
  • the overall size of the vector should be approximately 26 kb to approximately 38 kb, and preferably from about 26 kb to about 37 kb for efficient packaging. If the above-described elements occupy less than about 26 kb, then "stuffer" DNA should be added in an amount so that the total length of the concatamerized viral vector is approximately 26-38 kb, or preferably about 26-37 kb. Thus the absolute amount of stuffer DNA may vary from vector to vector depending on the size of other elements present, but will generally be up to about 26 kb in length. It is preferred that the stuffer DNA not be expressed by the host cell.
  • the stuffer DNA will be inserted between the first packaging signal (element B) and the first heterologous expression cassette (element C) and/or between the first or second expression cassette (elements C or F) and the second packaging signal sequence element (element D).
  • the stuffer DNA is inserted prior to the concatamerization event, in an amount approximately one-half of the desired final size, or up to about 13 kb per non-concatamerized virus. This will result in a concatamerized virus containing up to about 26 kb stuffer DNA.
  • stuffer DNA sequences include: fragments of the hypoxanthine guanine phosphoribosyl-transferase gene, and other non-expressed human or mouse DNA fragments, preferably comprising minimal or no repeat regions.
  • the first 5'ITR and packaging signal ( ⁇ ) will generally be a sequence of up to approximately 500 bp.
  • the adenoviral ITRs are known in the art. One such ITR is encompassed in the left end of Ad5, base pairs 1-360, although additional base pairs may be added without affecting the ITR and packaging signal function. 5' ITRs are generally preferred to 3' ITRs.
  • the expression cassette contains the gene of interest, along with control sequences (i.e. promoter, termination signals, enhancers, etc.). These may be virtually any known gene, known promoter, and known control sequences which are desired.
  • the viruses of this invention have many advantages over prior adenoviral vectors. Their structure is very stable during multiple propagations. While not wishing to be bound by theory, it appears that the combination of viral size and arrangement of packaging signals is an important feature of this invention, and leads to unexpectedly beneficial results. Repeated rescue results in an unexpectedly consistent single concatamerization species, the tail-to-tail dimer, so generation of these types of viruses are repeatable. Unlike previous literature reports describing heterogeneous populations of concatamerized adenoviruses, no other concatamerization species were identified in any of the three independent rescues (by restriction mapping and analysis of radio-labeled digestion fragments).
  • helper virus contamination load is consistently very low, less than 0.1% per infectious HD unit and less than 1 pfu of helper virus/100,000 OD particles (minimum estimated HD infectious unit: OD particle is 1 : 100).
  • OD particle is 1 : 100.
  • helper-dependent adenoviruses which comprises less than about 0.5% helper contamination or less than lpfu helper virus per 100,000 OD particles.
  • HD-leptin-monomer virus containing: a 5TTR and packaging signal sequence at the left arm only; stuffer DNA; the expression cassette; additional stuffer DNA; and the 3' ITR of Ad5; consistently results in at least 2-10 fold higher load of helper virus contamination (1 pfu of helper virus/103-104 OD particles/ml) in HD-leptin monomer stock.
  • a further advantage with the concatamerized virus according to this invention is the longevity of transgene expression achieved.
  • the leptin model used in these examples illustrates this advantage.
  • the transgene was expressed for a significantly longer time when introduced into the mouse using the 2 ⁇ -vector as compared to a non-concatamerized control adenoviral vector.
  • the concatamerized viruses of this invention expressed a transgene, leptin, at levels comparable to its counterpart first generation Ad-leptin.
  • leptin a transgene that has been modified by the concatamerized viruses of this invention.
  • HD-2 ⁇ - leptin One virus according to this invention has been designated HD-2 ⁇ - leptin. It was generated from a 16.7 kb vector fragment ( Figure 1). This fragment when transfected and propagated in the presence of a helper virus resulted in a concatamerized virus with a full length of approximately 33 kb. The full length structure of the tail-to-tail concatamerized recombinant virus is detailed in Figure IB.
  • mice either control lean or obese (ob/ob) were treated with a single tail intravenous infusion of 1-2 x 10l 1 particles of either: • HD-2 ⁇ -leptin ⁇ a concatamerized virus according to this invention; • Ad-leptin ⁇ a so-called first generation adenoviral vector, non-concatamerized, containing viral genes other than El, and a leptin transgene expression cassette;
  • control virus Ad- ⁇ -gal the same first generation adenoviral vector as Ad-leptin, except that the transgene is ⁇ -gal; • an equal volume of dialysis buffer control.
  • Liver toxicity As reflected by the significant elevation in AST and ALT serum levels over basal control levels, was observed only in mice treated with Ad- ⁇ -gal and Ad-leptin, but not HD-2 ⁇ -leptin.
  • Ad-vector-associated toxicity observed in both the lean and ob/ob treated mice was most significant at one week, was present but to a less significant extent at two weeks, and was resolved by four weeks post-treatment.
  • HD-2 ⁇ -treatment was not associated with liver toxicity as reflected by the AST and ALT serum levels which were essentially indistinguishable from controls.
  • both Ad- ⁇ -gal and Ad-leptin treated mice displayed degenerative hepatic pathology characterized by foci of round cell infiltration composed almost entirely (>98%) of T-cells, individual liver cell necrosis, increased liver cell mitotic activity, and dissociation of hepatic cords.
  • Ad-leptin treated mice displayed a similar, but less pronounced hepatic pathology.
  • Vector DNA in the livers of Ad-leptin treated mice was rapidly lost and fewer than 0.2 copies per cell were detected, compared to 1-2 copies per cell following HD- 2 ⁇ -leptin treatment at 8 weeks post-injection. These effects can be correlated with the duration of gene expression obtained with these two vector types.
  • Gene expression mediated by Ad-leptin was transient and almost undetectable as early as 1 week post- treatment as seen by northern blot analysis of total liver RNA, whereas that mediated by HD-2 ⁇ -leptin persisted for at least eight weeks. No changes in serum glucose or insulin levels in the treated lean mice were detected throughout the study. Vector DNA levels were stable at 1-2 copies per cell at 1, 2, 4 and 8 weeks post-treatment.
  • Leptin serum levels The ob/ob mice are naive to leptin and thus transgene immunogenicity is not an unexpected finding. In these animals, similar to what was observed in the lean mice, HD-2 ⁇ -leptin was found to be less immunogenic than the first-generation Ad-leptin vector. In the ob/ob mice treated with Ad-leptin, serum levels of leptin increased only for a short period during the first four days of treatment, returning to baseline levels within ten days post-injection. Increased leptin levels was associated with transient body weight loss of approximately 25%, followed by weight gain two weeks after treatment.
  • the Ad- leptin vector DNA was rapidly lost (less than 0.2 copies per cell were detected by 2 weeks post treatment, and undetectable by 8 weeks).
  • the ob/ob HD-2 ⁇ -leptin-treated mice had increased serum leptin levels up to approximately 15 days post-treatment, after which the levels gradually dropped to baseline over the subsequent 25 days.
  • the initial rise in leptin levels correlated with rapid weight reduction resulting in greater than 60% weight loss (reaching normal lean weight) by one month. Weight loss was maintained for a period of 6-7 weeks post-treatment.
  • As leptin levels dropped to baseline a gradual increase in body weight was observed. Satiety was observed in association with increased leptin levels, and appetite suppression was sustained for a longer period (approximately 1 month) compared to the short transient effect induced by Ad-leptin (approximately 10 days) (data not shown).
  • Leptin-specific antibodies were detected in the sera of ob/ob Ad-leptin- and HD-2 ⁇ -leptin -treated mice, therefore it was essential to determine whether the drop observed in serum leptin levels was due to interference of the antibodies with the ELISA assay utilized to measure leptin, or a loss of vector DNA and/or gene expression.
  • the analysis revealed eventual loss of the HD-2 ⁇ -leptin DNA over the 8 week time interval.
  • Serum glucose and insulin Serum glucose and insulin. Serum glucose and insulin levels dropped during the first 1-2 weeks post-treatment to normal lean values in both HD-2 ⁇ -leptin- and Ad-leptin- treated mice, although the effects of HD-2 ⁇ -leptin treatment were sustained for longer periods, which parallels what was seen with weight loss, satiety, DNA stability and leptin gene expression. The subsequent increase in glucose and insulin levels in both vector treatments correlated with the drop observed in serum leptin levels and eventual loss of vector DNA. The overall HD-2 ⁇ -leptin-mediated prolonged effect was also reflected in the accompanying phenotypic correction, which lasted longer than that seen in litter mates treated with Ad-leptin (6-7 versus 2-3 weeks).
  • leptin transgene cassette is just a single embodiment of this invention. Virtually any expression cassette containing any desired transgene may be substituted for the illustrated leptin cassette.
  • a non-concatamerized vector comprises the following elements (in 5' to 3' order): a) a first 5'-inverted terminal repeat sequence (ITR); b) a first packaging signal; c) one or more heterologous expression cassettes; d) a second packaging signal; and e) a second 5' ITR wherein the vector has an overall size of about 26 to about 38 kb, and wherein the only adenoviral sequences present in the vector are ITRs and packaging signals, and wherein the vector contains no bacterial plasmid-based sequences.
  • the non- concatamerized vector may contain "stuffer" DNA to increase its size.
  • the stuffer DNA may be placed anywhere in the vector, but is preferably placed between the first packaging signal and the hererologous expression unit (items b) and c)), and/or between expression units (c) or between the last expression unit and the second packaging signal (c) and d)).
  • the amount of stuffer DNA will depend on the size of the heterologous expression cassette(s), but will be enough to bring the total size of the vector to about 26 to about 38 kb.
  • these vectors contain non- bacterial plasmid based sequences.
  • the heterologous expression cassettes (element C) cannot be a bacterial gene—the heterologous expression cassette may contain any desired gene and control (promoter, enhancer, termination sequence), regardless of origin.
  • the vector does not contain extraneous sequences which are from the plasmid, however, such as a bacterial origin of replication, or marker genes which are soley useful in the plasmid culture of the vector.
  • these vectors can be purified so that a population of the rescued vectors contains a very low level of contaminating helper virus (less than about 0.1%), thus making them useful as vectors for human gene therapy.
  • HCMV promoter Invitrogen, Carlsbad, CA
  • the transgene either leptin or ⁇ -galacto
  • HD-2 ⁇ -leptin was prepared by releasing the linear backbone structure of HD-leptin from its plasmid p ⁇ STK120-HCMV-mOb-BGHpA (by Pmel digest) (described in co-pending U.S. application Serial No. 08/878,737 and WO 97/48806, published December 24, 1997, both of which are hereby incorporated by reference, and further described below) and transfecting the linear DNA into 293 -CRE cells followed by helper infection as described below.
  • HD-2 ⁇ -leptin plasmid is a pBluescriptlLKS based plasmid that contains (in the following order): a) the Ad5 inverted terminal repeat sequences (ITR) and the packaging signal ⁇ , 440 base pairs (bp), (nucleotides (nt.) 1-440); b) "stuffer DNA" which is a 5072 bp fragment of hypoxanthine guanine phosphoribosyl transferase (HPRT) (nt.12373 - 17781 in Genbank gb : humhprtb); c) the leptin gene expression cassette, 1835 bp including the HCMV promoter, the leptin cDNA and the bovine growth hormone polyadenylation signal sequence; d) a Hindlll 9063 bp fragment
  • the total size of this construct is 19.6 kb, which includes 2.9 kb of the pBluescriptllKS.
  • the 2.9 kb of pBluescriptllKS is eliminated prior to HD-vector rescue by linearizing the plasmid with two Pmel flanking sites.
  • HD-leptin-monomer plasmid (pSTK120-HCMV-mOb-BGHpA) differs in that the HPRT "stuffer DNA" is a larger fragment of 16054 bp (nt. 1799-17853 in Genbank gb:humhprtb).
  • Total size of the HD-leptin-monomer plasmid is approximately 30 kb, which includes 2.9 kb of the pBluescriptllKS, which as in the case of HD-2 ⁇ -leptin plasmid, is also eliminated by linearizating the plasmid with two Pmel flanking sites and releasing the HD-leptin-monomer fragment.
  • the HD-2 ⁇ -leptin vector DNA was excised from the plasmid backbone by Pmel digestion and 4 ⁇ g were used to transfect semi-confluent 293-cre4 cells in 6 cm plates. Following an overnight incubation, cells were infected at a multiplicity of infection (moi) of 1 with the helper virus AdLC8clucl.
  • CPE complete cytopathic effect
  • COS cells were infected with 10 ⁇ l of HD-2 ⁇ -leptin or with Ad-leptin at an moi of 10 or 15. Cells were washed 30 minutes post-infection and serum-free media was added. One-hundred-microliter aliquots of media were collected from infected plates at 24, 30, 48 and 54 hours post-treatment, and compared by western blot analysis for leptin protein levels.
  • the HD-2 ⁇ -leptin mediated expression was equivalent to the 15 moi-infected plates, and based on the plaque-forming-unit (pfu) titer of Ad-leptin, the estimated infectious titer was approximately 1-2 x 10 0/ml with a particle to infectious unit ratio of approximately 1 : 100.
  • the helper virus (AdLC ⁇ clucl) content in the HD-2 ⁇ -leptin stock was 1.5 x 107 pfulvcA.
  • Fifty microliters (1- 2 x l ⁇ H OD particles/dose, containing approximately 7.5 x 10$ pfu helper, i.e. less than 0.001% contamination with helper/ estimated infectious HD dose) of the stock were diluted with dialysis buffer to 100 ⁇ l for the mouse tail vein injections.
  • Digested viral DNA was analyzed by Southern blot analysis: fragments were radio-labeled using T4 DNA polymerase; DNA was run on a 1.0 or 0.5%» (for sizing purposes in case of undigested DNA extracted from HD-2 ⁇ -leptin and Ad-leptin) agarose gels in TAE buffer; gels were dried and exposed to auto- radiography for detection of radio-labeled, digested bands or stained with ethidium bromide and photographed (in case of undigested viral DNA). Fifty - 100 ng of purified viral DNA were used for each digestion.
  • Figure IB shows the structure and similarity of vectors according to this invention (33 kb), which are all tail-to-tail concatemerizations (junction is at the 3' ITR ends of ⁇ STK120-HCMV-mOb-BGHpA).
  • Vector DNA shows 0.5 ⁇ g of DNA extracted from the HD-2 ⁇ - leptin viral stock (Lane A), Ad-leptin viral stock (Lane B) and the Pmel cut p ⁇ STK120-HCMV-mOb-BGHpA viral stock (Lane C) compared on a 0.5%) agarose gel for sizing.
  • Both HD-2 ⁇ - leptin (33 kb) and Ad-leptin (34 kb) extracted DNA migrate, as expected, between 38.5 - 29.9 kb, and the cut ⁇ STK120- HCMV-mOb-BGHpA (16.7 kb) migrates between 17.1 and 15.0 kb, the smaller band corresponds to the plasmid backbone (2.9kb), and the faint band in lane A represents the trace amount of the propagated unconcatemerized HD-leptin (16.7 kb).
  • the expected fragment sizes for HD-2 ⁇ -leptin are: Asp718: 15391-single band (-s), 6296-double band (-d), and 2501-d; Eagl: 20445-s and 6270/6266-d;
  • Hindlll 10207/10174-d, 5845-d, and 454/450-d; Pad: 16516/16465-d;
  • the expected fragment sizes for ⁇ STK120-HCMV-mOb-BGHpA are: Asp718: 7837-s, 6296-s, and 2501-s; Eagl: 10364-s and 6266-s; Fsel: 16458-s and 172-s; Hindlll: 10174-s, 5848-s, 450-s and 158-s; Pacl: 16465-s and 165-s;
  • Xho 1 11833-s, 2964-s, 1697-s and 136-s bp.
  • Ml and M2 are DNA markers ( 8-48 Kb, Bio-Rad Laboratories, Hercules, CA and 1 kb DNA ladder, Life Technologies / GIBCO BRL, Gaithersburg, MD, respectively ). Detection of leptin protein expression mediated by the HD-leptin recom-binant virus in vitro. COS cells were infected at 15 and 10 multiplicities of infection (moi) of Ad- leptin, and HD-2 ⁇ - leptin with 10 ⁇ l of the viral stock.
  • moi multiplicities of infection
  • primer J4-F 5'- CTC TTC TTC TGT CAC ACC CCT CCC-3' (SEQ.ID.NO. 1) was used individually to amplify the junction-fragment of HD-leptin.
  • the fragment generated was approximately 300 bp, and was cloned into PCR 2.1 vector (Invitrogen, Carlsbad, CA) and sequenced.
  • HD-2 ⁇ -hVEGFl45 plasmid is a pBluescriptllKS based plasmid that contains (in the following order): a) the Ad5 inverted terminal repeat sequences (ITR) and the packaging signal ⁇ , 440 base pairs (bp), (nucleotides (nt.) 1-440); b) "stuffer DNA” which is a 16054 bp fragment of hypoxanthine guanine phosphoribosyl transferase (HPRT) (nt.
  • ITR Ad5 inverted terminal repeat sequences
  • bp base pairs
  • nt. nucleotides
  • HPRT hypoxanthine guanine phosphoribosyl transferase
  • hVEGF human vascular endothelial growth factor —145 amino acids
  • the total size of this construct is about 29 kb, which includes 2.9 kb of the pBluescriptllKS.
  • the 2.9 kb of pBluescriptllKS is eliminated prior to HD-vector rescue by linearizing the plasmid with two Pmel flanking sites.
  • the structure of the HD-l ⁇ -hVEGFl45 plasmid is identical to that of the HD-2 ⁇ -hVEGFl45 plasmid except for its right end. It lacks the second packaging signal, but contains the 3' ITR sequence, 117bp (nt. 35818-35935).
  • the plasmid also includes 2.9 kb of the pBluescriptllKS, which as in the case of HD-2 ⁇ -leptin plasmid, is also eliminated by linearizating the plasmid with two Pmel flanking sites and releasing the HD-vector.
  • EXAMPLE 6 Rescue and propagation of HD vectors with 1 or 2 packaging signals (1 or 2 ⁇ ) Plasmids were transfected into 293 cre4 cells constitutively expressing the ere recombinase using the Calcium phoshate coprecipitation technique. Cells were infected with a helper virus (El -deleted vector with lox sites flanking the packaging signals, AdLC8BHG10lucl) 36 h after transfection. Both viruses were continuously passaged three times in 6 cm plates followed by 15 cm plates 3 times by infecting 293- cre4 cells with 1 ml (6 cm plates) or 5 ml (15 cm plates) cell lysate from the previous passage and the helper virus (1 plaque forming unit (pfu) per cell).
  • helper virus El -deleted vector with lox sites flanking the packaging signals, AdLC8BHG10lucl
  • HD-vector preparations were further analyzed.
  • Productivity was 4.3 times higher for the vector containing 2 ⁇ . 6.9 x 104 particles/cell of the HD hVEGFl45 2 ⁇ were produced.
  • the contamination of the HD-vectors by helper virus was determined by quantitative PCR using Taqman Technology. Helper contamination was 5-fold lower for the vector containing 2 ⁇ (contamination 0.4%). Helper contamination was further reduced to 0.2% after three more passages.
  • mice In Vivo Studies Mouse colony, ob/ob (C57BL/J6-ob/ob) mice and homozygous normal lean (C57BL/J6) litter mates (age-matched females), were purchased from Jackson Laboratories (Bar Harbor, ME) for use in this study. Animals were free of all common murine pathogens. Eight-to twelve-week-old mice (ob/ob approximately 70 g and lean approximately 28 g) were re-distributed based on equal representation of weight and caged in groups of five on day 0, immediately preceding treatment.
  • mice After a series of baseline blood samples were obtained by tail incision from conscious mice, animals were divided into four groups and received by tail vein injection a single 100 ⁇ l aliquot containing 1-2 XlOl particles of HD-2 ⁇ -leptin, Ad-leptin, Ad- ⁇ -gal (control), or dialysis buffer (control). Body weight and food intake were measured daily and blood was collected 2-3 times weekly, pre- and post-treatment. Animals were killed by carbon dioxide inhalation and organs removed for immuno-histochemistry and RNA analysis. All animals used in this study were maintained in accordance with the "Guide for the Care Use of Laboratory Animals" (DHHS Publication No.(NIH) 85-23, revised 1996). The protocol was approved by the Institutional Animal Care and Use Committee, Merck Research Laboratories, West Point, PA.
  • T cell T cell
  • CD45R B cell
  • Plasma samples were obtained by tail incision and collected into heparinized microhematocrit tubes (VWR) every 2-3 days during the course of the study. Tubes were centrifuged at 13,700 g for 2 minutes and hematocrit values were monitored. Plasma was collected for measurement of aspartate aminotransferase (AST), alanine amino-transferase (ALT), leptin, glucose and insulin levels. ALT and AST were measured by using the ALT/SGPT and AST/SGOT DT slides, respectively (Vitros Chemistry Products, Johnson & Johnson Clinical Diagnostics Inc., Rochester, NY). Leptin and insulin levels were measured by a radio-immunoassay performed by Linco Research, Inc. Glucose levels were measured using the Kodak Ektachem DT slides (Eastman Kodak Com.).
  • Northern and Southern blot analysis For northern blot analysis, total RNA was extracted (Trizol, Gibco) from livers of Ad-leptin-treated and HD-leptin-2-treated mice at 1-, 2-, 4-, and 8-week intervals, and untreated mice. Leptin message was detected by northern blot analysis (Maniatis et al, 1982 Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) using leptin cDNA as a probe which recognizes a single approximately 500 bp band Morsy et al, 1997 supra. A probe for ⁇ -actin was used as the internal control (approximately 1 kb) (Biochain Inc.). Southern blot analysis (Maniatis et al, 1982 supra) was used to investigate the stability of vector DNA.
  • control DNA 20 ⁇ g control DNA were spiked with HD vector DNA equivalent to 2.0, 1.0, 0.2 and 0.1 copies per cell, and the mixture digested with Hindlll restriction enzyme followed by Southern blotting.
  • the filters were hybridized with a mouse leptin cDNA (approximately 500 bp) probe, which hybridized to a single Hindlll fragment containing the leptin insert in both the HD-2 ⁇ - leptin (approximately 6 kb), and Ad-leptin (approximately 1.2 kb) vectors.
  • This invention is related to improved adenoviral vectors which are useful as delivery systems in gene therapy. Specifically, it has been found in accordance with this invention that an adenoviral vector comprising a packaging signal sequence at either end has improved characteristics for its propagation and use as a vector.
  • a helper-dependent (HD) adenoviral stock comprising less than about 0.5% helper virus contamination, wherein the HD adenovirus is devoid of adenoviral DNA except for inverted terminal repeat and packaging signal sequences.
  • the HD adenoviral stock is free from detectable levels of helper virus contamination.
  • Another embodiment of this invention is a concatamerized vector comprising the following elements (in 5' to 3' order): a first 5 '-inverted terminal repeat sequence (ITR), a first packaging signal, a first heterologous expression cassette, a first 3' ITR an optional second 3' ITR, a second heterologous expression cassette, a second packaging signal and a second 5' ITR and which has an overall size of about 26 to about 38 kb, and wherein the only adenoviral sequences present in the vector are ITRs and packaging signals.
  • ITR 5 '-inverted terminal repeat sequence
  • Another embodiment of this invention is a single copy vector comprising the following elements (in 5' to 3' order): a first 5'-inverted terminal repeat sequence (ITR), a first packaging signal, one or more heterologous expression cassettes, a second packaging signal and a second 5 'ITR and which has an overall size of about 26 to about 38 kb, wherein the only adenoviral sequences present in the vector are ITRs and packaging signals, and wherein there are no bacterial plasmid based sequences (such as an origin of replication or bacterial marker genes) present in the adenoviral vector.
  • ITR 5'-inverted terminal repeat sequence
  • viral vectors of this invention will be referred to as HD-2 ⁇ vectors.
  • the vectors may comprise additional, non-expressed portions of DNA, termed "stuffer DNA", which may be inserted at one or more locations between the above-recited elements.
  • shuffer DNA additional, non-expressed portions of DNA
  • a vector containing only a heterologous expression cassette, adenoviral ITR and packaging signals may be quite small, in general about 5-10 kb, depending on the size of the heterologous expression cassette. Such a small virus does not package efficiently. Even if duplicated, the virus may be still too small to package efficiently. However, if the size of the vector is more than about 12 kb and vector is duplicated to generate an HD virus of an overall sized of more than about 25 kb, then efficient packaging can occur.
  • Stuffer DNA thus may be any DNA used to increase the size of a vector. It has been found, in accordance with this invention, that populations of concatamerized viruses are remarkably homogeneous, and importantly, have a very low level of helper virus contamination. Thus, another aspect of this invention is a population of concatamerized adenoviral vectors which comprises less than about 0.1% per infectious HD unit and less than 1 pfu of helper virus/100, 000 OD particles (minimum estimated HD infectious unit: OD particle is 1 : 100).
  • HD vectors require more passages than first generation adenoviruses until a stock is suitable for purification and characterization.
  • At early passages only a small percentage of cells is infected with the HD vector. Because the remaining cells are still infected with helper virus in the absence a packageable HD genome, these cells are likely to generate packagable variants of the helper.
  • Vectors with the described features (2 ⁇ -vectors and concatamerized vectors) reach high titer already at passage 5 or 6, thereby minimizing the risk of helper variants.

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Abstract

L'invention porte sur des vecteurs adénoviraux dépendant des cellules auxiliaires et pouvant d'utiliser en thérapie génique du fait de leur stabilité, de leur très faible contenu (moins de 0,5%) de virus auxiliaires contaminants, et de leur caractère moins immunogène que les vecteurs adénoviraux antérieurs. Les seuls ADN viraux des vecteurs sont des signaux ITR et d'emballage et leur longueur totale est comprise entre environ 26 et 38 kb.
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