JP2002517234A - Novel adenovirus vectors for gene therapy - Google Patents

Abstract

  (57) [Summary] Helper-dependent adenovirus vectors are suitable for use in gene therapy because they are stable, have minimal helper virus contamination of less than about 0.5%, and are less immunogenic than existing adenovirus vectors. I have. Viral DNA in the vector is limited to ITRs and packaging signals only, total size is about 26-38 kb.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The present invention relates to an adenovirus vector useful as a vector for gene delivery in gene therapy applications.

BACKGROUND Adenoviruses (Ad) vectors of the present invention are currently is one of the either the most efficient gene transfer vehicle in vitro and in vivo delivery, many gene therapy current Ad vectors There are limits to their use in applications because of the transient expression of the transgene obtained with these vectors. With the development of Ad vectors in which the coding sequence of the viral protein has been completely deleted, the possibility of a safer and less immunogenic vector having an insert capacity of at most about 38 kb is expected. These vectors require in-trans capture of the viral regulatory and structural proteins required for packaging and excision,
These vectors are called "helper dependent" (HD).

[0003] Fisher et al., 1996, Virology 217: 11-22 describe the spontaneous concatemerization of HD-adenovirus vectors. This results in three concatemerized viruses with different genome sizes, namely type 1 having the genome size of the original transfected DNA, type 2 having twice the size of the monomer, and a larger predominant type 3 A population containing Individual genomes are fused in three different ways: a “head-to-tail” structure (5′-3′-5′-3 ′, ie, 5 ′ and 3 ′ regions at both ends). Included, with the 3 'region connected to the 5' region in the center), a "head-to-head" structure (5'-3'-3
'-5', that is, 5 'regions at both ends and two 3' regions joined at the center
), As well as a `` tail-to-tail '' structure (3'-5'-5'-3 ', i.e., containing 3' regions at both ends,
Two 5 'regions are connected in the middle). These viruses exist as multimeric mixtures of small genomes, and adenovirus sequences are limited to only the ITR and packaging regions.

[0004] Parks et al., 1997, J. Virology 71 (4): 3293-3298 discloses that a 15.1-kb virus spontaneously concatamerizes in culture to form two different 30.2 kb dimers. A population of adenovirus has been described. Parks describes the structure of the following concatemers using a different terminology than Fisher: the "head-to-tail" dimer (5'-3'-5'-3
', I.e., the 5' region is included at one end, the 3 'region is included at the other end, and the 3' region is linked to the 5 'region in the middle), as well as the dimer is "tail- Another concatemer that is a "tail" dimer (ie, contains a 3 'region at both ends, with the 5' regions joined in the middle). These vectors contain large segments of the adenovirus genome, such as the disrupted E1A gene (+5,700 bp in the 5 ′ region and +6,100 bp in the 3 ′ region).
Area).

[0005] Kochanek et al., 1996, Proc. Natl. Acad. Sci. USA 93: 5731-5736 states that as a result of extensive deletions, the viral sequences present are necessary for the packaging of DNA into viral capsids. Terminal repeats (ITRs) and full-length packaging signals (
An adenovirus vector limited to only ψ) is described. These vectors are capable of carrying large heterologous DNA inserts. However,
In the vector there is also DNA from the plasmid used for replication before the excision step (eg ori and ampicillin resistance genes). Such vectors cannot avoid high levels of contamination (about 1%) with helper virus, which levels are too high for human gene therapy applications.

[0006] One of the problems associated with HD-adenovirus as a vector for gene therapy is that the HD-virus population is contaminated with helper viruses necessary for virus culture and replication. That is why it is desirable to obtain an HD-system with low helper virus contamination.

[0007] The following definitions are used herein and in the claims: stuffer DNA: inserted into an adenovirus vector to reduce the size of the virus to at least about 75% of wild-type, more preferably wild-type. At least about 75 of the mold
Non-expressing DNA that increases by ~ 105% (about 26-38 kb).

[0008] HD-virus: A "helper-dependent" virus: An adenovirus that lacks a native gene and cannot replicate unless the product of the deleted gene is supplied by another source or cell line, such as a helper virus. Expression cassette: contains the gene to express the product and all necessary regulatory sequences. Tail-to-tail concatemerization: the region of 5'-3'-3 after joining two viruses
It becomes '-5'. Head-to-tail concatemerization: the region of 5'-3'-5 after joining two viruses
It becomes '-3'. Head-to-head concatemerization: the region of 3'-5'-5 is obtained by linking two viruses.
It becomes '-3'.

[0009] Plasmid-based sequence: DNA present in a plasmid that is used in the construction of the adenovirus vector but is neither the adenovirus DNA nor the heterologous gene to be delivered by the adenovirus vector And a gene. Examples of plasmid-derived sequences include an origin of replication (ori), an antibiotic resistance gene used as a marker (ampicillin resistance gene), and the like.

[0010] Adenovirus vectors are generally useful for delivering genes in gene therapy. Previously, adenovirus vectors containing deletions in the genomic portion encoding functions involved in replication, such as the E1 gene and E3 gene, have been produced. These deletions have two different functions. That is, not only is a safe non-replicating viral vector produced, but the deletion also leaves room for insertion of the transgene. Such viruses with replication deficiencies are usually helper-dependent (H
D)-Called virus. Such viruses cannot replicate on their own, but co-culture of helper virus with HD-
can be supplied in s.

[0011] Propagation of the HD-virus using a normal cell line and a helper virus containing genes required for virus replication resulted in spontaneous concatemerization. These new HD-2ψ-viruses were excised, propagated, and analyzed for both structural confirmation and helper load contamination. Alternatively, these viruses can also be prepared by ligating the various described segments using ordinary genetic engineering techniques.

The concatamerized HD-adenovirus vector of the invention comprises the following elements: (A) a first 5′-inverted terminal repeat (ITR), (B) a first packaging signal sequence. An element, (C) a first heterologous expression cassette, (D) a second packaging signal sequence element, and (E) a second 5'ITR.

Further, the vector may comprise 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 cassettes may express the same gene or may express different gene products.

The total size of the vector ranges from about 26 kb to about 38 kb for efficient packaging,
Preferably it should be about 26 kb to about 37 kb. If the above-described elements are less than about 26 kb, "stuffer" DNA must be added in an amount such that the total length of the concatemerized viral vector is about 26-38 kb, 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 is usually up to about 26 kb in length. Preferably, the stuffer DNA is not expressed by the host cell. Generally, the stuffer DNA is between the first packaging signal (element B) and the first heterologous expression cassette (element C) and / or the first or second expression cassette (element C).
Insert between element C or F) and the second packaging signal sequence element (element D). In a preferred embodiment, the stuffer DNA is inserted at about half the desired final size or up to about 13 kb per non-concatamerized virus prior to the concatamerization event. This allows for stuffer DNA up to about 26kb
A concatemerized virus containing Examples of suitable stuffer DNA sequences include fragments of the hypoxanthine guanine phosphoribosyltransferase gene and other non-expressing human or mouse DNA fragments, preferably those that contain minimal or no repetitive sequences. No.

The first 5′ITR and the packaging signal (ψ) are usually a sequence of up to about 500 bp. Adenovirus ITRs are known in the art. One such ITR is
Included at the left end of Ad5 (base pairs 1-360), additional base pairs can be added so as not to affect the function of the ITR and the packaging signal. 5'ITR is usually more preferred than 3'ITR.

The expression cassette contains a gene of interest as well as regulatory sequences (ie, a promoter, termination signal, enhancer, etc.). These can be virtually any known gene, known promoter, and known control sequences, as desired.

[0017] The viruses of the present invention are superior in many respects to existing adenovirus vectors. Its structure is very stable during growth. Without wishing to be bound by theory, it is believed that the combination of the size of the virus and the placement of the packaging signal is an important feature of the present invention and provides unexpected and beneficial results. The generation of these types of viruses is repetitive, since repeated excision unexpectedly produces a single concatenated species, the tail-to-tail dimer. Previous literature reports described a heterogeneous population of concatemerized adenoviruses, whereas 3
No other concatamerized species was identified in any of the independent cuts (by restriction mapping and restriction analysis of radiolabeled digested fragments).

A further significance of the virus of the invention is that the load of contamination by helper virus is consistently very low, ie less than 0.1% of helper virus per HD infectious unit and less than 1 pfu per 100,000 OD particles. Yes (HD infectious unit: estimated minimum value of OD particles is 1: 100). Thus, one aspect of the invention is a population of helper-dependent adenoviruses that has less than about 0.5% helper contamination or less than 1 pfu of helper virus per 100,00 OD particles.

In contrast, the 5'ITR and packaging signal sequence located only on the left arm,
Stuffer DNA, expression cassette, additional stuffer DNA, and 3'ITR of Ad5
HD-leptin monomer virus consistently results in a load of at least 2 to 10 times higher helper virus contamination in the HD-leptin monomer strain (1 pfu helper virus / 10 ³ -10 ⁴ OD particles / ml). ).

A further advantage associated with the concatamerized virus of the present invention is that the life span of transgene expression is extended. The leptin model used in the examples below illustrates this advantage. When the transgene was introduced into mice using the 2ψ-vector, the transgene was expressed for a significantly longer period than the non-concatemerized control adenovirus vector.

The concatamerized virus of the present invention expressed the transgene leptin at a level comparable to its corresponding first generation Ad-leptin. Therefore, when the virus of the present invention is used, expression can be maintained for a long period of time, and it is possible to maintain the same expression level during this expression period.

One of the viruses of the present invention was named HD-2ψ-leptin. The virus is 16.
It was prepared from a 7 kb vector fragment (FIG. 1). Transfection and propagation of this fragment in the presence of a helper virus yielded a concatamerized virus with a full length of about 33 kb. The full length structure of the tail-to-tail concatamerized recombinant virus is detailed in FIG. 1B.

Mice (both control lean and ob / ob) were given 1-2 × 1
0 ¹¹ particles were treated by a single intravenous injection: HD-2ψ-leptin-the concatamerized virus of the present invention-Ad-leptin-a so-called first generation adenoviral vector other than E1 A non-concatenated vector containing a viral gene and a leptin transgene expression cassette; a control virus Ad-β-gal --- a first generation adenoviral vector similar to Ad-leptin except that the transgene is β-gal Hepatotoxicity, represented by a significant increase in AST and ALT serum levels relative to equivalent dialysis buffer control hepatotoxicity control levels, was observed only in mice treated with Ad-β-gal and Ad-leptin, HD -2
It was not found in mice treated with ψ-leptin. The toxicity associated with the Ad-vector observed in both lean and ob / ob treated mice was most pronounced at week 1,
It was observed at the second week but was not remarkable and disappeared by four weeks after the treatment. In contrast, HD-2ψ-treatment is essentially indistinguishable from controls and has no hepatotoxicity as represented by AST and ALT serum levels.

Liver sections from lean mice treated with HD-2ψ-leptin were histologically compared to control liver sections at any time after the treatment (1 week, 2 weeks and 4 weeks). Did not differ. A spontaneous perivascular cluster consisting of 50:50 T and B cells and small foci of cell infiltrates were observed in HD-2ψ-leptin treated mice and untreated control mice. In contrast, Ad-leptin and Ad-β-gal treated mice consistently showed liver lesions after treatment. One week after treatment, both Ad-β-gal and Ad-leptin-treated mice had almost all (> 98%) round cell infiltrates consisting of T cells, individual hepatocyte necrosis, and hepatocyte mitotic activity. It showed degenerative liver lesions characterized by enhancement and separation of hepatocytes. Two weeks after treatment, Ad-leptin-treated mice showed similar liver lesions but were clearly reduced. Observed inflammation and cell infiltration disappeared by 4 weeks after treatment, with few lesions in Ad-leptin-treated mice, with only evidence of individual cell death falling within the normal range .
Examination of liver sections obtained from ob / ob mice showed similar tissue lesions associated with the Ad-vector. Similar to observations in lean mice, no evidence of toxicity associated with the Ad-vector was observed with HD-2ψ-leptin treatment in ob / ob mice, but some cell infiltration was detected. This cell infiltration may be due to leptin immunogenicity in these leptin-deficient mice. Nevertheless, the degree of inflammation and cell infiltration was at significantly lower levels than those observed with Ad-leptin.

Transgene expression period In lean mice, treatment with Ad-leptin resulted in a temporary increase in serum leptin levels, and weight loss persisted for only 7-10 days. In contrast, treatment with HD-2ψ-leptin resulted in higher serum leptin levels (6-10 times background) and approximately 20% weight loss persisted for at least 2 months. Weight loss in HD-2ψ-leptin-treated mice is related to satiety, satiety in Ad-leptin-treated mice (5-7 days)
Longer (2-3 weeks). Vector D was used in the liver of Ad-leptin-treated mice.
NA disappeared rapidly and only less than 0.2 copies of vector per cell were detected compared to 1-2 copies per cell at 8 weeks post injection in cells after HD-2ψ-leptin treatment. Such effects may be correlated with the duration of expression of the genes obtained with these two vector types. Gene expression mediated by Ad-leptin is transient and is almost undetectable as early as one week after treatment, as evident from Northern blot analysis of total liver RNA, whereas it is mediated by HD-2ψ-leptin. The resulting gene expression lasted for at least 8 weeks. Lean treated mice showed no change in serum glucose or insulin levels during the study. Vector DNA levels were stable at 1-2 copies per cell at 1, 2, 4 and 8 weeks after treatment.

Leptin serum levels Ob / ob mice have never been affected by leptin, so the immunogenicity of the transgene is not an unexpected finding. In such animals, HD-2ψ-leptin was found to be less immunogenic than the first generation Ad-leptin vector, as observed in lean mice. In ob / ob mice treated with Ad-Leptin, serum levels of leptin rose only for a short period during the first 4 days of treatment and returned to baseline levels within 10 days after injection. Elevated leptin levels were associated with a temporary loss of body weight of about 25% followed by an increase two weeks after treatment. Similar to the results obtained in lean mice, Ad-leptin vector DNA disappeared rapidly (less than 0.2 copies per cell were detected up to 2 weeks after treatment and became undetectable by 8 weeks ).

In contrast, in ob / ob HD-2ψ-leptin treated mice, serum leptin levels increased for up to about 15 days after treatment and then gradually decreased to baseline over 25 days. Early increases in leptin levels correlate with rapid weight loss,
By one month caused more than 60% weight loss (reaching normal lean weight).
Weight loss lasted 6-7 weeks after treatment. As leptin levels fell to baseline, a modest increase in body weight was observed. Satiety was observed with increasing leptin levels, and the short-term transient effects induced by Ad-leptin (approximately 10
Appetite suppression lasted longer (approximately 1 month) than in the case of day (data not shown).

Leptin-specific antibodies were detected in the sera of ob / ob Ad-leptin-treated mice and ob / ob HD-2ψ-leptin-treated mice. Thus, it was determined whether the observed decrease in serum leptin levels was due to interference of the ELISA assay used to measure leptin with the antibody or to the loss of vector DNA and / or gene expression. Judgment was essential. Southern blot analysis showed that in the livers of ob / ob treated mice, HD-2ψ-leptin DNA was more stable than Ad-vector DNA when compared at similar time points, This analysis revealed that HD-2ψ-leptin DNA eventually disappeared during eight weeks. In the liver of the HD-2ψ-leptin-treated mice, 4 weeks and 8 weeks after treatment, compared to the sustained levels seen in the liver of HD-2HD-leptin-treated lean littermates at similar time points.
About 75% less vector DNA was detected per week. Gene expression in ob / ob Ad-leptin-treated mice correlated with DNA findings, with RNA levels deviating from the level of detection sensitivity one week after treatment, whereas in HD-2ψ-leptin-treated mice, Gene expression was detected up to 4 weeks after injection and became undetectable at 8 weeks.

Serum Glucose and Insulin Serum glucose levels and serum insulin levels were normal in both HD-2ψ-leptin-treated mice and Ad-leptin-treated mice during the first 1-2 weeks after treatment. However, the effect of HD-2ψ-leptin treatment lasted longer. This was also the case for weight loss, satiety, DNA stability and leptin gene expression. Glucose and insulin levels subsequently increased upon treatment with both vectors, which was related to the observed decrease in serum leptin levels and eventual loss of vector DNA. All of the long-acting effects mediated by HD-2ψ-leptin were also reflected in the accompanying phenotypic correlation and were longer lasting than those seen in littermates treated with Ad-leptin (6-7). Weeks versus 2-3 weeks).

Our studies revealed that HD-2ψ-leptin significantly improved the safety profile and gene expression longevity over the first generation Ad-leptin vector. . Significant differences observed in the degree of cellular invasion in the liver, significant hepatotoxicity associated with Ad-leptin treatment in lean mice (AST and ALT serum levels increased by about 10- and 5-fold), and HD- The absence of them in 2ψ-leptin treatment may be directly attributable to the removal of the DNA sequence encoding the adenovirus protein, as long as the leptin expression cassette is identical in both vectors. Appearance of leptin-specific antibody response, i.e., slow gene expression and vector DNA observed in ob / ob (leptin-deficient) mice treated with HD-2ψ-leptin but not observed in lean mice (leptin-wild type) The major loss reflects an independent immune response event involved in leptin resistance.

It should be understood that the use of a leptin transgene cassette is only one embodiment of the present invention. Virtually any expression cassette containing any desired transgene can be replaced with the exemplary leptin cassette.

In another embodiment of the invention, the non-concatemerizing vector comprises: a) a first 5′-inverted terminal repeat (ITR), b) a first packaging signal, c) one or more of A heterologous expression cassette, d) a second packaging signal, and e) a second 5′ITR element in the 5 ′ to 3 ′ direction, said vector having a total size of about 26
~ 38 kb, the adenovirus sequences present in the vector are limited to ITRs and packaging signals only, and the vector does not contain bacterial plasmid-derived sequences.

Like the concatemerized vectors described above, the non-concatamerized vector may contain “stuffer” DNA for the purpose of increasing the amount. In this vector, the stuffer DNA can be placed at any position in the vector, but preferably,
Between the first packaging signal and the heterologous expression unit [between elements b and c],
And / or within the expression unit (c) or between the final expression unit and the second packaging signal [between c and d]. The amount of stuffer DNA depends on the size of the heterologous expression cassette, but it is sufficient if the total size of the vector is about 26 to about 38 kb.

Another important feature of these vectors is that they do not contain sequences derived from bacterial plasmids. This does not mean that the heterologous expression cassette [element c] must not be a bacterial gene. The heterologous expression cassette can contain any desired gene and control sequences (promoter, enhancer, termination sequence), regardless of origin. The vector does not include a plasmid-derived foreign sequence, even if it is a bacterial origin of replication or a marker gene useful only in plasmid culture of the vector.

Because these vectors can be purified, the excised population of vectors contains only very low levels of contaminating helper virus (less than about 0.1%), and is therefore Was found to be useful as a vector. Hereinafter, the present invention will be further described with reference to examples, but the present invention is not limited to these examples.

[0036] The construction of the first generation vector was named as in Example 1 Vector Construction of "Ad- leptin" and "Ad-β-gal",
Essentially, Morsy et al., 1998, Gene Therapy 5 (1): 8-18, which is hereby incorporated by reference. Briefly, the expression cassette contains an HCMV promoter (Invitrogen, Carlsbad, CA), a transgene (either leptin or β-galactosidase), and a bovine growth hormone polyA sequence. These vectors are described in Graham et al., 1997 J. Gen. Virol. 36: 59-72 and Graham et al., 19
91 Gene Transfer and Expression Protocols, Murray, EJ (The Humana Pr
Proliferation and titration were performed as described in ess Inc., Clinton, NJ), pp. 109-128. Both documents are incorporated herein by reference.

HD-2ψ-leptin is obtained by combining the linear backbone structure of HD-leptin with its plasmid pΔSTK120-HCMV-mOb-BGHpA (co-pending US patent application Ser. No. 08 / 878,737 and 1997). Described in WO 97/48806, published December 24; both are incorporated herein by reference and are further described below), and are released by digestion with PmeI and linear DNA into 293-CRE cells. It was prepared by transfecting and carrying out helper infection.

[0038] Two different structures, HD-2ψ-leptin and HD-leptin monomer, were used for excision of HD viruses expressing leptin. The structure of the HD-2ψ-leptin plasmid is as follows: a) Ad5 inverted terminal repeat (ITR) and packaging signal ψ (440 base pairs (bp)
) (Nucleotides 1-440), b) 5072 bp of hypoxanthine guanine phosphoribosyltransferase (HPRT)
The fragment `` Stuffer DNA '' (nucleotides 12373-177 of Genbank gb: humhprtb)
81), c) leptin gene expression cassette (1835 bp) containing HCMV promoter, leptin cDNA and bovine growth hormone polyadenylation signal sequence, d) HindIII 9063 bp fragment of C346 cosmid (Genbank gb: nucleotides 1242 of L31948)
1-21484), and e) a pBluescript IIKS-based plasmid containing, in this order, the right end (117 bp) (nucleotides 35818-35935) of Ad5 consisting of a 3′ITR sequence having a multiple cloning site interposed at the junction of different fragments. is there.

The total size of this construct is 19.6 kb, including 2.9 kb of pBluescriptIIKS. 2.9k
pBluescriptIIKS in b, before excision of the HD-vector,
Remove linearly at the meI flanking site.

The HD-leptin monomer plasmid (pSTK120-HCMV-mOb-BGHpA) differs in that the HPRT “stuffer DNA” is a large fragment of 16054 bp (nucleotide 1799-17853 of Genbank gb: humhprtb). The total size of the HD-leptin monomer plasmid is about 30 kb, including 2.9 kb of pBluescriptIIKS. The 2.9 kb pBluescriptIIKS is removed by linearizing the plasmid with two PmeI flanking sites and releasing the HD-leptin monomer fragment, as in the case of the HD-2ψ-leptin plasmid.

Example 2 Propagation of HD-Virus Propagation of both HD-viruses has a lox site adjacent to the packaging signal
Helper virus system carrying the E1 deletion vector [AdLC8clucl; Parks et al., 1996, Pr
USA 93: 13565-13570 (incorporated herein by reference)] and a derivative line of the 293 cell line expressing Cre recombinase [293-cre4; Lie
ber et al., 1996, J. Virol. 70: 8944-8960 (incorporated herein by reference) and Parks, RJ et al., 1996, supra]. HD-2ψ-leptin vector DNA was excised from the plasmid backbone by PmeI digestion and 4 μg was used to transfect semi-confluent 293-cre4 cells in a 6 cm plate. After overnight incubation, cells were infected with the helper virus AdLC8clucl at a multiplicity of infection (moi) of 1.

At the time of harvesting the cells, the complete cytopathic effect (CPE) of the cells was monitored and the lysate was analyzed in Parks et al., 1996, supra and Parks et al., 1997, J. Virol. 71: 3293-3298. It was used for continuous propagation of the virus strain according to the modification described. Briefly, 2 ml of lysate recovered from P1 (transfection / infection stage) was used to infect a semi-confluent 293-cre4 6 cm plate for 24 hours (supplemented with 1 ml of fresh medium). . After incubation for 24 hours, the helper virus AdLC8clucl was added dropwise to the cells at a multiplicity of infection of 1. The P2 lysate was collected when CPE was detected. The same procedure was repeated continuously for another three growths (P3, P4 and P5),
A 10 cm plate of 293-cre4 cells at semi-confluence and then a 15 cm plate were infected. Using the lysate recovered from P4, infect 20 15 cm plates (2
1 ml of lysate was added to 4 ml of fresh medium), and the lysate was banded with cesium chloride as previously described in Graham et al., 1991, supra. The isolated virus was analyzed by restriction mapping and the HD-2ψ-leptin virus was sequenced for structure confirmation.

The final strain of HD-2ψ-leptin was reduced to about 1.2 × 10⁹Cells from 293-cre4 cells
When isolated with calcium, the yield of the virus strain was about 8 × 10¹²Particles (2 × 10¹²/ ml)
there were. For semi-quantitative analysis of infectious particles, 10 μl of HD-2ψ-leptin or Ad-
Leptin was infected into COS cells at a multiplicity of infection of 10 or 15. 30 minutes after infection of cells
After washing, serum-free medium was added. 100 μl aliquot of medium from infected plate
Collected at 24, 30, 48 and 54 hours after treatment, and leptin by Western blot analysis
Comparisons were made for protein levels. HD-2ψ-leptin-mediated expression
Is equivalent to a plate infected at a multiplicity of infection of 15, and the Ad-leptin
The estimated infectious titer, based on the chromogenic unit titer (pfu), is the ratio of particle to infectious unit.
Rate is about 1: 100 and about 1-2 × 10^Ten/ ml. Helperw in HD-2ψ-leptin strain
ILS (AdLC8clucl) content is 1.5 × 10⁷ pfu / ml. 50 μl of the strain (about 7.5 × 10 ^Five 1-2 x 10 including pfu helper¹¹ OD particles / dose, ie,
Less than 0.001% of helper contamination) with dialysis buffer for tail vein injection in mice.
Diluted to 100 μl.

Example 3 Repetitive excision of HD-2ψ-leptin virus The excision of HD-leptin recombinant virus was independently performed three times. First stage (P1) pΔ
Starting with STK120-HCMV-mOb-BGHpA transfection,
A similar stable structure of leptin was obtained. Asp718, EagI, FseI, HindIII, PacI, Sma
Seven different enzymes, I and XhoI, were used to confirm the viral structure. The digested viral DNA was analyzed by Southern blot analysis. That is, the fragments are radiolabeled using T4 DNA polymerase, and the DNA is 1.0 or 0.5% (when separating undigested DNA extracted from HD-2ψ-leptin and Ad-leptin) on agarose gel in TAE buffer. The gels were dried and autoradiographed to detect digested radiolabeled bands, or stained with ethidium bromide and photographed (for undigested viral DNA). Each digestion was performed using 50-100 ng of purified viral DNA.

FIG. 1B shows the structure and similarity of the vector (33 kb) of the present invention. Such vectors form a tail-to-tail concatemer (ligated at the 3′ITR end of ΔSTK120-HCMV-mOb-BGHpA). In FIG. 2, the gels labeled “Vector DNA” are HD-2ψ-leptin virus strain (lane A), Ad-leptin virus strain (lane B) and PmeI-cut pΔ, respectively.
0.5 μg of DNA extracted from STK120-HCMV-mOb-BGHpA virus strain (lane C) was
% Agarose gel separated and compared. Both HD-2ψ-leptin (33 kb) and Ad-leptin (34 kb) extracted from DNA migrated between 38.5 and 29.9 kb, as expected, and the cleaved ΔSTK120-HCMV-mOb-BGHpA (16.7 kb) was 17.1 kb. Runs between 15.0 kb. The smaller band corresponds to the plasmid backbone (2.9 kb), and the faint band seen in lane A corresponds to a trace of unconcatenated HD-leptin (16.7 kb) that has grown.

In the remaining gel, ΔSTK120-HCMV-mOb-BGHpA (lane 1) (the plasmid backbone was replaced with PmeI
The structures of the gels extracted after separation by digestion) and the three HD-2ψ-leptin vectors (lanes 2, 3 and 4) are compared by restriction analysis as described in the examples.

The estimated fragment size (bp) of HD-2ψ-leptin is as follows: Asp718: 15391-single band (-s), 6296-double band (-d), and 2501-d; EagI : 20445-s and 6270 / 6266-d; FseI: 16523 / 16458-d; HindIII: 10207 / 10174-d, 5845-d and 454 / 450-d; PacI: 16516 / 16465-d; SmaI: 6701-d , 5163-d, 2180-d, 1715-s and 1589-d; XhoI: 11833-d, 2964 / 2953-d and 1701 / 1697-d.

The estimated fragment sizes (bp) of ΔSTK120-HCMV-mOb-BGHpA are as follows: Asp718: 7837-s, 6296-s and 2501-s; EagI: 10364-s and 6266-s; FseI: 16458-s and 172-s; HindIII: 10174-s, 5848-s, 450-s and 158-s; PacI: 16465-s and 165-s; SmaI: 6701-s, 5163-s, 2180-s, 1589-s and 997-s; XhoI: 11833-s, 2964-s, 1697-s and 136-s. M1 and M2 are DNA markers (8-48 kb, respectively, Bio-Rad Laboratories
, Hercules, CA and 1kb DNA Ladder, Life Technologies / GIBCO BRL, Gaither
sburg, MD).

Leptin protein mediated by HD-leptin recombinant virus in vitro
Detection of quality expression At multiplicity of infection (moi) of 15 and 10, Ad-leptin and HD-2ψ-leptin were used to infect COS cells with 10 μl of the virus strain. Uninfected control cells (C-) and 24, 30, 48 post-infection
The medium was collected from the cells at 54 and 54 hours (100 μl), and the secreted leptin was examined by Western blotting using a polyclonal anti-leptin antibody (Santa Cruz Biotech.). Leptin was detected as a single band (about 16 kD).

Example 4 PCR Amplification of the Connected Fragments and Sequencing Primers adjacent to the connected fragments were used for PCR. That is, primer J4-F: 5 ′
The ligation fragment of HD-leptin was amplified using -CTC TTC TTC TGT CAC ACC CCT CCC-3 '(SEQ ID NO: 1). The resulting fragment is approximately 300 bp and contains a PCR 2.1 vector (Invitro
gen, Carlsbad, CA) and sequenced.

The structure of the building HD-2ψ-hVEGF ¹⁴⁵ plasmid having different HD virus of Example 5 packaging signal, a) inverted terminal repeats of Ad5 (ITR) and a packaging signal [psi (440 base pairs ( bp)
) (Nucleotides 1-440), b) 16054b of hypoxanthine guanine phosphoribosyltransferase (HPRT)
`` Stuffer DNA '' which is a p-fragment (nucleotides 1799-178 of Genbank gb: humhprtb)
53), c) Expression cassette (1301 bp) of the gene encoding hVEGF (human vascular endothelial growth factor, 145 amino acids), including the HCMV promoter, cDNA and SV40 early polyadenylation signal sequence, d) HindIII 9063 bp fragment of C346 cosmid ( Genbank gb: nucleotide 1242 of L31948
1-21484), and e) a second packaging signal followed by a second left terminal ITR sequence (440 base pairs (bp)) with multiple cloning sites intervening at the junction of the different fragments (nucleotides 1- 440) in this order.

The total size of this construct is about 29 kb, including 2.9 kb of pBluescriptIIKS. this
The 2.9 kb pBluescriptIIKS contains 2 plasmids prior to excision of the HD-vector.
Remove linearly at two PmeI flanking sites.

The structure of the HD-1ψ-hVEGF ¹⁴⁵ plasmid is identical to the structure of the HD-2ψ-hVEGF ¹⁴⁵ plasmid except for the right end. The HD-1ψ-hVEGF ¹⁴⁵ plasmid lacks the second packaging signal but contains the 3′ITR sequence (117 bp, nucleotides 35818-35935)
. This plasmid also contains 2.9 kb of pBluescriptIIKS. This 2.9kb pBlue
ScriptIIKS is removed by linearizing the plasmid with two PmeI flanking sites and releasing the HD-vector, as in the case of the HD-2ψ-leptin plasmid.

Example 6 HD vector carrying one or two packaging signals (1 (or 2ψ)
The excised and expanded plasmid was transfected into 293-cre4 cells constitutively expressing cre recombinase using the calcium phosphate co-precipitation method. 36 hours after transfection, the helper virus (E1- with a lox site adjacent to the packaging signal)
Cells were infected with the deletion vector, AdLC8BHG10lucl). Both viruses were serially passaged three times on 6 cm plates and then three times on 15 cm plates. For passage, 1 ml (for a 6 cm plate) or 5 ml (for a 15 cm plate) of the cell lysate obtained from the previous passage and helper virus (1 plaque forming unit (pfu) per cell) were added to 293-cre4.
This was done by infecting cells. Propagation of both viruses was performed in parallel.

Cells passaged six times (two 15 cm plates) were harvested and lysed, and the lysate was banded with cesium chloride as previously described in Graham et al., 1991, supra. HD-vector and helper were not separated during purification. DNA was extracted from the separated virus and analyzed by restriction mapping using the restriction enzyme HindIII. Fragments were labeled with Klenow enzyme and electrophoresed, and the gel was exposed to film (FIG. 4A). The restriction pattern of both HD-vectors matches the expected pattern. 1 contamination with helper DNA
Note that this is only confirmed in the case of a vector containing a single packaging signal.

[0056] The HD-vector preparation was further analyzed. The amount of HD-vector particles produced per cell was determined by measuring the optical density (OD) at 260 nm. It was converted using the formula of 1 OD = 1.1 × 10 ¹² particles.

Productivity was 4.3-fold higher for vectors containing 2ψ. 6.9 × 10 ⁴ particles of HD hVEGF ¹⁴⁵ 2ψ per cell were produced. Contamination of the HD-vector by helper virus was measured by quantitative PCR using Taqman Technology. Helper pollution is 2
In the case of the vector containing ψ, the ratio was 1/5 (contamination rate: 0.4%). After three additional passages, helper contamination was further reduced to 0.2%.

Example 7 In Vivo Study Mouse colony ob / ob (C57BL / J6-ob / ob) mice and homozygous normal lean (C57BL / J6) littermates (females of the same age) were used for this study. Purchased from Jackson Laboratories (Bar Harbor, ME).
Animals were not infected with any of the normal mouse pathogens. Mice aged 8 to 12 weeks (ob / ob: about 70 g, lean type: about 28 g) were re-classified by body weight and transferred to cages immediately before treatment on day 0 in groups of 5 mice. After collecting a series of reference blood samples by tail incision from non-anesthetized mice, the animals were divided into four groups and 1-2 × 10 ¹¹ particles of HD-2ψ-
Contains leptin, Ad-leptin, Ad-β-gal (control), or dialysis buffer (control)
A single 100 μl aliquot was injected into the tail vein. Body weight and food intake were measured daily before and after treatment, and blood was collected 2-3 times weekly. Animals were sacrificed by carbon dioxide inhalation and organs were removed for immunohistochemistry and RNA analysis. All animals used in this study were referred to as `` Guide for the Care Use of Laboratory Animals '' (DHHS issue number (
NIH) 85-23, revised 1996). This protocol is based on Institutiona
l Animal Care and Use Committee, Merck Research Laboratories, West Point
, PA approved.

Histopathological Studies Mice (three at one time per treatment) were euthanized and liver samples were collected and fixed in 10% buffered formalin. Tissues were treated with paraffin as usual, sectioned at 5 microns, and stained with hematoxylin and eosin. In addition, prepare a similar unstained slide for immunohistochemistry using a standard method, and use it on invaded cells or original cells.
Staining was performed for the presence of CD3 determinants (T cells) and CD45R determinants (B cells) (data not shown).

Blood Measurements Blood samples were collected by tail incision every 2-3 days during the study and collected in heparinized microhematocrit tubes (VWR). The tubes were centrifuged at 13,700 g for 2 minutes and the hematocrit was monitored. Plasma was collected and measured for aspartate aminotransferase (AST), alanine aminotransferase (ALT), leptin, glucose and insulin levels.
ALT and AST were measured using ALT / SGPT DT slides and AST / SGOT DT slides, respectively (Vitros Chemistry Products, Johnson & Johnson Clinical Diagnostics I
nc., Rochester, NY). Leptin and insulin levels are measured by Linco Research,
Measured by radioimmunoassay performed at Inc. Glucose levels were measured using Kodak Ektachem DT slides (Eastman Kodak Com.).

Northern and Southern Blot Analysis For Northern blot analysis, total RNA was extracted from livers of Ad-leptin-treated and HD-leptin-2 treated mice at 1, 2, 4 and 8 week intervals (Trizol). , Gi
bco). Total RNA was also extracted from untreated mice. Northern blot analysis using leptin cDNA (Morsy et al., 1997, supra) as a probe that recognizes a single band of about 500 bp (Maniatis et al., 1982, Molecular Cloning: A Laboratory Manual Co.
ld Spring Harbor Laboratory, Cold Spring Harbor, NY), the genetic code of leptin was detected. A probe for β-actin (about 1 kb) was used as an internal control (
Biochain Inc.). The stability of the vector DNA was examined using Southern blot analysis (Maniatis et al., 1982, supra).

Genomic DNA was extracted from the livers of ob / ob mice and lean mice treated with Ad-vector, ie, Ad-β-gal, Ad-leptin or HD-2ψ leptin (pool DNA, 1).
3 animals per time point per treatment). Control animals were injected with the same volume of dialysis buffer.
The pooled genomic DNA (for 3 animals) was digested with HindIII restriction enzyme, and 20 μg of the digested DNA was added to 0.1 μl.
Loaded on 8% TAE agarose gel.

For copy number estimation, 20 μg of control DNA was used at 2.0, 1.0, 0.2 and 0.1 per cell.
Spiking was performed with HD vector DNA corresponding to the copy, and the mixture was digested with HindIII restriction enzyme and subjected to Southern blotting. Mouse leptin cDNA probe for membrane
(About 500 bp), both the HD-2ψ-leptin vector (about 6 kb) and the Ad-leptin vector (about 1.2 kb) hybridized with a single HindIII fragment containing a leptin insert. Scan the developed autoradiograph (Pe
rsonal Densitometer SI, Molecular Dynamics, Sunnyvale, CA) and relative band density was quantified (Image QuaNT software, Molecular Dynamics). To estimate the relative stability of vector DNA between treatment time points at each time point, an internal leptin genomic DNA signal, which is also used in normalizing DNA loading, was detected and used as a control. A value corresponding to the copy number was assigned by comparing the relative density ratio between the internal genomic signal and the leptin signal of the spiked vector DNA in the copy number control experiment.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to improvements in adenovirus vectors useful as delivery systems in gene therapy. Specifically, according to the present invention, it has been found that an adenovirus vector comprising a packaging signal sequence at each end has improved growth characteristics and characteristics relating to use as a vector. One embodiment of the present invention is a helper-dependent (HD) adenovirus strain that has less than about 0.5% helper virus contamination, the HD adenovirus containing an inverted terminal repeat and a packaging signal sequence. Adenovirus DNA is defective. In another embodiment, the HD adenovirus strain has a helper virus contamination below the limit of detection.

[0065] Another embodiment of the present invention is directed to a first 5'- inverted terminal repeat (ITR), a first packaging signal, a first heterologous expression cassette, a first 3'ITR, optionally Second three
'ITR, second heterologous expression cassette, second packaging signal and second 5'
A concatemerization vector comprising 'ITR elements in the 5' to 3 'direction, the total size of which is about 26 to about 38 kb, and the adenovirus sequence present in the vector contains only the ITR and the packaging signal. It is limited.

Another embodiment of the present invention is directed to a first 5′-inverted terminal repeat (ITR), a first packaging signal, one or more heterologous expression cassettes, a second packaging signal, and A single copy vector comprising a second 5′ITR element in the 5 ′ to 3 ′ direction, having a total size of about 26 to about 38 kb, wherein the adenoviral sequences present in the vector are ITR It is limited to only the packaging signal, and does not include bacterial plasmid-derived sequences such as an origin of replication or a bacterial marker gene present in the adenovirus vector. In the present specification and claims, the virus vector of the present invention will be referred to as HD-2ψ vector.

In addition to the above elements, the vector may further comprise a non-expressed portion of DNA (referred to as “stuffer DNA”), and can be inserted at one or more positions between the above elements. Vectors containing only the heterologous expression cassette, adenovirus ITR and packaging signal are very small, usually about 5-10 kb, depending on the size of the heterologous expression cassette. Such small viruses do not provide efficient packaging. Even if replicated, such viruses may still be too small for efficient packaging. However, if the size of the vector is increased to about 12 kb or more, the vector can be replicated to generate an HD virus having a total size of about 25 kb or more, and efficient packaging can be performed. Also, if non-expressing "stuffer" DNA is added to the vector, its final size will be at least about 75% of wild-type adenovirus, and efficient packaging will occur. Thus, the stuffer DNA can be any DNA used to increase the size of the vector.

According to the present invention, it has been found that the population of concatamerized viruses is significantly homogeneous and, importantly, the level of helper virus contamination is very low. Thus, another embodiment of the present invention provides that the helper virus is less than about 0.1% per HD infectious unit;
And a population of concatemerized adenovirus vectors that are less than 1 pfu per 100,000 OD particles (HD infectious units: estimated minimum of OD particles 1: 100).

Another aspect of the invention is to promote the accumulation of proliferating HD vectors early in the passage. HD vectors require further passage from the first generation adenovirus until a strain is suitable for purification and characterization. Early in the passage, only a small portion of the cells is infected with the HD vector. These cells may generate packageable variants of the helper, since the remaining cells would still be infected with the helper virus in the absence of the packageable HD genome.
Vectors having the above characteristics (2ψ-vectors and concatemerized vectors)
High titers are already reached in 5 or 6 passages, minimizing the risk of helper mutants.

[Brief description of the drawings]

FIG. 1 is a diagram of HD-leptin monomer and HD-2ψ-leptin as vectors. Figure
1A is a DNA complex fragment of pΔSTK120-HCMV-mOb-BGHpA (total size is about 19.6 kb).
This fragment, from left to right, contains the inverted terminal repeat and the packaging signal 左側, the left end of Ad5 (nucleotides 1-440, filled arrow); a 5072 bp fragment of hypoxanthine guanine phosphoribosyltransferase (HPRT) (Genbank g
b: nucleotides 12373-17853 of humhprtb, vertical lines) (inserted in complementary orientation); HindIII 9063 bp fragment of C346 cosmid (nucleotides 12421-21484 of gb: L31948)
, Marked part); and the right end of Ad5 consisting of the ITR sequence (nucleotides 35818-
35935). The ITR is flanked by a unique PmeI restriction site that releases the vector fragment from the plasmid backbone prior to transfection into 293-cre4 cells for virus excision and propagation. Used for (
The free fragment is 16.7 kb). FIG. 1B shows HD-2ψ-leptin, a virus of the present invention.
This is a tail-to-tail concatamerization of the virus of FIG. 1A.

FIG. 2 is a series of gels showing the size of the vector prepared according to the present invention and the size of the vector cut with various restriction enzymes. Details are described in Examples.

Figure 3 is a vector (A) stk120-1ψ-CMV-hVEGF 145 -SV40pA and (B) stk120-2ψ
It is a diagram of -CMV-hVEGF ¹⁴⁵ -SV40pA. The fragment of FIG. 3 (A) is, from left to right, the left end of Ad5 (nucleotide containing the inverted terminal repeat and packaging signal ヌ ク レ オ チ ド).
1-440, filled arrow); 16054 bp fragment of hypoxanthine guanine phosphoribosyltransferase (HPRT) (nucleotides 12373-17853 of Genbank gb: humhprtb)
, Vertical line) (inserted in complementary orientation); HindIII 9063 bp fragment of C346 cosmid (gb: nucleotides 12421-21484 of L31948, marked part); and right end of Ad5 consisting of ITR sequence ( Nucleotides 35818-35935). The fragment of (B) is (A)
Except that the right end of Ad5 has been replaced with an inverted left end and contains the inverted terminal repeat and the packaging signal ψ (nucleotides 1-440, filled arrows). The ITR is flanked by a unique PmeI restriction site that is used to release vector fragments from the plasmid backbone prior to transfection into 293-cre4 cells for virus excision and propagation. (Free fragment 29 kb).

FIG. 4 shows a comparison between vector HD-1ψ and vector HD-2ψ after propagation. (A) Restriction analysis of DNA isolated from the HD-vector preparation at passage 6. HD-vector and helper were not separated during purification. Restriction fragments were radiolabeled and separated by electrophoresis, and the gel was exposed to film. The estimated size of the restriction fragments for both HD-vectors is then shown on the autoradiograph. The band (450 bp) containing the second packaging signal of the 2ψ vector overlaps with the 475 bp band, and the unique band (145 bp) of the right ITR cannot be confirmed in this autoradiograph. The restriction pattern of the helper virus is shown for comparison. (B) Characteristics of the 6th passage HD vector preparation: Productivity was determined by the optical density (OD) of purified HD-vector particles obtained per cell from the 6th passage preparation, and the purity was determined. Measured the percent contamination of the HD-vector with helper virus by quantitative PCR using Taqman Technology.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), CA, JP, US (72) Inventor Sandik, Volker United States 07065 New Jersey, Lowway, East Lincoln Avenue 126

Claims (19)

[Claims] Claims 1. A helper dependency, wherein helper virus contamination is less than about 0.5%.
HD) An adenovirus population, wherein each HD adenovirus contained in the population lacks adenovirus DNA other than the inverted terminal repeat sequence and the packaging sequence. 2. A helper-dependent (HD) adenovirus vector comprising: (A) a first 5'-inverted terminal repeat (ITR); (B) a first packaging signal sequence element; ) A first heterologous DNA and a heterologous expression cassette, (D) a second packaging signal sequence element, and (E) a second 5'ITR element, wherein said vector has a total size of about 26 to about 38kb,
The vector, wherein the adenovirus sequences present in the vector are limited to ITRs and packaging signals only. 3. The second heterologous expression cassette (F) is replaced with the first heterologous expression cassette (C).
3. The vector according to claim 2, further comprising between the second packaging signal (element D). 4. The vector according to claim 2, further comprising stuffer DNA. 5. The vector of claim 4, wherein the first heterologous expression cassette is substantially identical to the second heterologous expression cassette. 6. The first heterogeneous expression cassette comprises a first transgene and the second heterologous expression cassette comprises a second transgene, wherein the first transgene and the second transgene are combined. 5. The vector of claim 4, wherein the genes are not the same transgene. 7. The vector according to claim 3, wherein the first and second heterologous expression cassettes comprise a leptin gene. 8. A concatemerized adenovirus vector comprising: (A) a first 5'-inverted terminal repeat (ITR); (B) a first packaging signal element; (C) a first heterologous. DNA and heterologous expression cassette, (D) first 3′ITR, (E) optionally second 3′ITR, (F) second heterologous DNA and heterologous expression cassette, (G) second packaging signal And (H) the element of the second 5′ITR in the 5 ′ to 3 ′ direction, wherein the vector has a total size of about 26
The above vector, further comprising a sufficient amount of stuffer DNA to be about 38 kb, wherein the adenovirus sequences present in the vector are limited to only the ITR and the packaging signal. 9. A host cell comprising the vector according to claim 1. 10. A population of helper-dependent adenoviruses, wherein the helper-dependent virus strain has less than 1 pfu of helper virus per 100,000 OD particles. 11. A single copy adenovirus vector, comprising: (A) a first 5'-inverted terminal repeat (ITR); (B) a first packaging signal; (C) one or more of: A heterologous DNA expression cassette, (D) a second packaging signal, and (E) a second 5′ITR element in the 5 ′ to 3 ′ direction, said vector having a total size of about 26
~ 38 kb, wherein the adenovirus sequences present in the vector are limited to ITRs and packaging signals only and do not include bacterial plasmid-derived sequences. 12. An element C comprising a first heterologous DNA expression cassette and a second heterologous DN.
The vector according to claim 11, comprising an A expression cassette. 13. The vector according to claim 11, further comprising stuffer DNA. 14. The vector of claim 12, wherein the first heterologous expression cassette is substantially identical to the second heterologous expression cassette. 15. The first heterogeneous expression cassette comprises a first transgene and the second heterologous expression cassette comprises a second transgene, wherein the first transgene and the second transgene. 13. The vector of claim 12, wherein the genes are not the same transgene. 16. The vector of claim 14, wherein said first and second heterologous expression cassettes comprise a human vascular endothelial growth factor gene. 17. A host cell comprising the vector according to claim 11. 18. A method for introducing a transgene into an animal, comprising introducing the adenovirus vector population of claim 1 into the animal. 19. The method of claim 18, wherein the animal is a human.