JP4683682B2 - Gene delivery vector imparted tissue tropism to smooth muscle cells and / or endothelial cells - Google Patents

Abstract

The invention provides a nucleic acid delivery vehicle with or having been provided with at least a tissue tropism for smooth muscle cells and/or endothelial cells. In one aspect said nucleic acid delivery vehicle is a virus capsid or a functional part, derivative and/or analogue thereof. Preferably said virus capsid is an adnovirus capsid. Preferably said adenovirus is a subgroup B adenovirus, preferably adenovirus 16. Preferably said tissue tropism is provided by at least a tissue tropism determining part of an adenovirus fiber protein or a functional derivative and/or analogue thereof. The invention further presents methods for the treatment of diseases, preferably cardiovascular diseases.

Description

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［参照文献］
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【配列表】

【図面の簡単な説明】
【図１】 pBr/Ad.Bam-rITR構築物の概略図である。
【図２】 pBr/Ad.Bam-rITR構築物からファイバー遺伝子を欠失させるために使用した方法の概略図である。
【図３】構築物pBr/Ad.BamRΔFibの概略図である。
【図４Ａ】ファイバーAd5の配列である。
【図４Ｂ】キメラファイバーAd5/12の配列である。
【図４Ｃ】キメラファイバーAd5/16の配列である。
【図４Ｄ】キメラファイバーAd5/28の配列である。
【図４Ｅ】キメラファイバーAd5/40-Lの配列である。
【図５】構築物pC1ipsal-Lucの概略図である。
【図６】３つの重複断片を使ってキメラアデノウイルスを作製する方法の概略図。初期領域（Ｅ）と後期領域（Ｌ）を示す。L5はファイバーコード配列である。
【図７ａ】 A)１細胞につき異なる量のウイルス粒子と異なるファイバーキメラアデノウイルスを用いたHUVEC細胞の感染。ウイルス濃度：10000vp/細胞（＝白い棒）、5000 vp/細胞（＝灰色の棒）、2500vp/細胞（＝黒い棒）、1000vp/細胞（薄い灰色の棒）、250および50vp/細胞はバックグランドを超える検出可能なルシフェラーゼ活性なし。ルシフェラーゼ活性は細胞タンパク質１マイクログラムあたりの相対発光単位（RLU）で表す。
【図７ｂ】 B)異なる濃度の細胞（１ウェルにつき22500、45000、90000または135000細胞を播種）とアデノウイルス血清型５（黒い棒）またはファイバー16キメラアデノウイルス（白い棒）を使ったHUVEC細胞の感染。ルシフェラーゼ活性は細胞タンパク質１マイクログラムあたりの相対発光単位（RLU）で表す。
【図７ｃ】 C)１細胞あたり500ウイルス粒子（黒い棒）または5000ウイルス粒子（灰色の棒）のAd5またはファイバー16キメラウイルス（Fib16）で形質導入したヒト大動脈ECのフローサイトメトリーによる分析。非感染細胞を使用して、バックグラウンドを１％、5.4の中央蛍光値に設定した。フローサイトメーターで観察できる中央蛍光値の最大シフトは9999である。後者は、Ad5とFib16が共に5000vp/細胞でフローサイトメーターの感度スケール外であることを示す。
【図８ａ】 A)１細胞につき異なる量のウイルス粒子と異なるファイバー突然変異型Ad5系アデノウイルスを用いたHUVsmc細胞の感染。ウイルス濃度：5000vp/細胞（＝白い棒）、2500vp/細胞（＝灰色の棒）、1250vp/細胞（＝濃い灰色の棒）、250vp/細胞（＝黒い棒）または50vp/細胞（＝薄い灰色の棒）。ルシフェラーゼ活性は細胞タンパク質１マイクログラムあたりの相対発光単位（RLU）で表す。
【図８ｂ】 B)異なる濃度の細胞（１ウェルにつき10000、20000、40000、60000または80000細胞）とアデノウイルス血清型５（白い棒）またはファイバー16キメラアデノウイルス（黒い棒）を使ったHUVsmc細胞の感染。導入遺伝子の発現が使用した生物発光計の感度域より高いという事実により、キメラファイバー16アデノウイルスによる感染後に頭打ちが観察される。
【図８ｃ】 C)Ad5またはファイバー16突然変異体（Fib16）を使用して500vp/細胞（黒い棒）または5000vp/細胞（灰色の棒）で形質導入したヒト臍静脈SMC。非形質導入細胞を使用して、バックグラウンド中央蛍光値を約１に設定した。フローサイトメーターによって測定されるGFP発現の中央蛍光値を示す。
【図８ｄ】 D)HUVsmcを１細胞につき312（薄い灰色の棒）、625（灰色の棒）、1250（黒い棒）、2500（濃い灰色の棒）、5000（薄い灰色の棒）または10000（白い棒）のウイルス粒子のファイバー11、16、35または51キメラウイルスに感染させた。タンパク質１マイクログラムあたりの相対発光単位（RLU）として表されるルシフェラーゼ導入遺伝子発現を、ウイルスばく露の48時間後に測定した。
【図８ｅ】 E)伏在静脈標本でのLacZ染色像の巨視的写真のスケッチ。核標的LacZ（ntLacZ）は深い青色、白黒印刷では黒または濃い灰色に見える。
【図８ｆ】 F)心膜（pericard）標本でのLacZ染色像の巨視的写真。核標的LacZ（ntLacZ）は深い青色、白黒印刷では黒色に見える。
【図８ｇ】 G)右冠状動脈標本でのLacZ染色像の巨視的写真のスケッチ。核標的LacZ（ntLacZ）は深い青色、白黒印刷では黒く見える。
【図８ｈ】 H)左下行動脈（Left artery descending；LAD）標本でのLacZ染色のスケッチ。核標的LacZ（ntLacZ）は深い青色、白黒印刷では黒色に見える。
【図９Ａ】ジーンバンク（Genbank）に公表されたアデノウイルス16ファイバータンパク質をコードする遺伝子を含む配列と、本発明で分離されたアデノウイルス16変異種のファイバーをコードする遺伝子を含む配列であり、ファイバータンパク質の配列はNdeI部位からである。図９Ａヌクレオチド配列比較。
【図９Ｂ】図９Ｂもヌクレオチド配列比較。
【図９Ｃ】アミノ酸比較。[0001]
Field of Invention
The present invention relates to the fields of molecular genetics and medicine. Specifically, the present invention relates to the field of gene therapy, more specifically gene therapy using adenovirus.
[0002]
Background of the Invention
Gene therapy usually involves genetic information in a host cell to correct (complement) a genetic defect in the cell, to suppress an undesirable function in the cell, or to eliminate the host cell. Is delivered. Of course, the genetic information is also intended to confer a desired function to the host cell, such as supplying a secreted protein to treat other cells in the host.
[0003]
Many different methods have been developed to introduce new genetic information into cells. There are many different systems that can work on cell lines cultured in vitro, but only a group of viral vector-mediated gene delivery methods can meet the required gene transfer efficiency in vivo. . Thus, for gene therapy purposes, most attention is directed to the development of suitable viral vectors. Currently, most attention for developing suitable viral vectors is directed to adenovirus-based vectors. These adenoviral vectors can deliver exogenous genetic information to target cells in vivo very efficiently. Furthermore, obtaining large quantities of adenoviral vectors is not a problem for most types of adenoviral vectors. Adenovirus vectors can be concentrated and purified relatively easily. Furthermore, clinical trial studies have provided valuable information regarding the use of these vectors in patients.
[0004]
There are many reasons for using adenoviral vectors to deliver nucleic acids to target cells in gene therapy protocols. However, some features of current vectors limit their use for specific applications. For example, endothelial cells and smooth muscle cells are not easily transduced with current generation adenoviral vectors. For many gene therapy applications, such as cardiovascular applications, these types of cells should preferably be genetically modified. On the other hand, for some applications, even the very good in vivo delivery capabilities of adenoviral vectors are insufficient and higher transfection efficiency is required. This is the case, for example, when it is necessary to transduce most cells of the target tissue.
[0005]
The present invention was carried out in the process of adenoviral vector manipulation. Therefore, the following section gives a brief overview of adenovirus.
[0006]
Adenovirus
Adenovirus contains a linear double-stranded DNA molecule of about 36000 base pairs. It contains about 90-140 base pairs of identical inverted terminal repeats (ITR), the exact length of which depends on the serotype. The viral origin of replication is at the very end of the genome within the ITR. The transfer unit is divided into an initial region and a late region. Shortly after infection, E1A and E1B proteins are expressed and function to transactivate cellular and adenoviral genes. The early regions E2A and E2B encode proteins required for replication of the adenovirus genome (DNA binding protein, pre-terminal protein and polymerase) (reviewed in 1995 van der Vliet). The early region E4 has multiple functions such as transactivation of the E2 early promoter, facilitating viral mRNA transport and accumulation in the late phase of infection, and increasing nuclear stability of the major late mRNA precursors. It encodes several proteins (reviewed in Leppard 1997). Early region 3 encodes a protein involved in the regulation of the host immune response (Wold et al., 1995). The late region is transcribed from a single promoter (major late promoter) and activated to initiate DNA replication. Complex splicing and polyadenylation mechanisms enable more than 12 RNA species to encode core proteins, capsid proteins (pentons, hexons, fibers and related proteins), viral proteases, proteins required for capsid assembly and host protein translation termination Resulting ("The molecular repertoire of adenoviruses I", edited by W. Doerfler and P. Bohm, Springer-Verlag, Berlin Heidelberg) Pp. 139-171, Imperialia, MJ, Akusjnarvi, G. and Leppard, KN (1995) “Post-transcriptional regulation of adenoviral gene expression (Post- transcriptional control of adenovirus gene expression) ").
[0007]
Interaction between virus and host cell
Virus interaction with host cells has been studied primarily with serotype C viruses Ad2 and Ad5. Binding occurs by interaction of the protruding fiber knob region with the cellular receptor. Ad2 and Ad5, and possibly more adenoviral receptors, are known as “Coxsackie virus and adenoviral receptors”, or CAR proteins (Bergelson et al., 1997). Internalization is mediated by the interaction of RGD sequences present in the penton base with cellular integrins (Wickham et al., 1993). This is not true for all serotypes, for example serotypes 40 and 41 do not contain an RGD sequence in their penton base sequence (Kidd et al., 1993).
[0008]
Fiber protein
The first stage of successful infection is adenovirus binding to target cells, a process mediated by fiber proteins. Fiber proteins have a trimeric structure (Stouten et al., 1992), and their length depends on the virus serotype (Signas et al., 1985; Kidd et al., 1993). Different serotypes have polypeptides that are structurally similar at the N and C terminus but differ in the intermediate stem region. The first 30 amino acids at the N-terminus are involved in anchoring the fiber to the penton base, particularly the conserved FNPVYP region (Arnberg et al., 1997) (Chroboczek et al., 1995). The C-terminus, or knob, is responsible for the initial interaction with the cellular adenovirus receptor. After this initial binding, secondary binding between the capsid penton base and the cell surface integrin causes viral particle internalization and endocytosis in the coated pit (Morgan et al., 1969; Svensson ) And Persson, 1984; Varga et al., 1991; Greber et al., 1993; Wickham et al., 1993). Integrins are αβ heterodimers in which at least 14 α subunits and 8 β subunits have been identified (Hynes, 1992). The set of integrins expressed in cells is complex and will vary from cell type to cell environment. The knob contains several conserved regions, but the knob protein shows a high degree of variability between serotypes, suggesting the presence of different adenoviral receptors.
[0009]
Adenovirus serotype
Currently, six different subgroups have been proposed for human adenovirus, including a total of about 50 adenovirus serotypes. In addition to these human adenoviruses, a number of animal adenoviruses have been identified (see, for example, Ishibashi and Yasue, 1984). A serotype is defined on the basis of its immunological discrimination determined by quantitative neutralization with animal antisera (horse, rabbit). If neutralization shows a certain degree of cross-reactivity between the two viruses, then A) those hemagglutinins are irrelevant as indicated by the lack of cross-reactivity in hemagglutination inhibition, or B) in the DNA If there is a substantial biophysical / biochemical difference, serotype discrimination is assumed (Francki et al., 1991). The last identified serotype (42-49) was first isolated from an HIV-infected patient (Hierholzer et al., 1988; Schnurr et al., 1993). For reasons that are not well understood, the majority of these immunocompromised patients release adenoviruses that were never isolated from immunocompetent individuals (Hierholzer et al., 1988, 1992; Khoo et al., 1995).
[0010]
In addition to differences in the susceptibility of various adenovirus serotypes to neutralizing antibodies, subgroup C adenoviruses such as Ad2 and Ad5 bind to different receptors compared to adenoviruses belonging to subgroup B such as Ad3 and Ad7. (Defer et al., 1990; Gall et al., 1996). Receptor specificity can also be altered by exchanging Ad3 knob protein for Ad5 knob protein, and vice versa (Krasnykh et al., 1996; Stevenson et al., 1995). Year, 1997). Serotypes 2, 4, 5, and 7 all have a natural close relationship with lung epithelium and other respiratory tissues. In contrast, serotypes 40 and 41 have a close natural relationship with the gastrointestinal tract. These serotypes differ at least in the capsid protein (penton-base, hexon), the protein responsible for cell binding (fiber protein), and the protein involved in adenovirus replication. It is not known how much the capsid protein determines the tropic difference seen between serotypes. It is quite likely that the mechanism after infection determines the cell type specificity of adenovirus. All adenoviruses belonging to serotypes A (Ad12 and Ad31), C (Ad2 and Ad5), D (Ad9 and Ad15), E (Ad4) and F (Ad41) can be labeled when immobilized on nitrocellulose. It has been shown that soluble CAR (sCAR) protein can be bound. Furthermore, the binding of adenoviruses belonging to these serotypes to Ramos cells (Roelvink et al., 1996) that express high levels of CAR but lack integrins causes the sCAR to become viral prior to infection. Can be efficiently blocked (Roelvink et al., 1998). However, the fact that (at least some) members of these subgroups can bind CAR does not exclude that these viruses have different infection efficiencies in different cell types. For example, subgroup D serotypes have a relatively short fiber shaft compared to subgroup A and subgroup C viruses. It has been speculated that the tropism of D subgroup viruses is largely determined by penton-based binding to integrins (Roelvink et al., 1996; Roelvink et al., 1998). Another example is that in 1998, 14 serotypes were examined for infection of human ciliary airway epithelium (CAE), and serotype 17 (D subgroup) is any other virus that contains other members of the D subgroup. Provided by Zabner et al., Who found that they are more efficiently combined and internalized. Similar experiments with primary fetal rat cells using serotypes belonging to the A to F subgroups show that adenoviruses belonging to subgroups A and B are ineffective, and viruses belonging to subgroup D are most effective. (Law et al., 1998). Again, viruses within one subgroup showed different efficiencies. The importance of fiber binding for the improved infectivity of Ad17 in CAE is that a recombinant LacZ Ad2 virus with an Ad17-derived fiber gene is produced and the chimeric virus is more efficient than a LacZ Ad2 virus with Ad2 fiber. Armentano et al. (International Publication No. WO98 / 22609) have been shown to be infected.
[0011]
Thus, despite the common ability of them to bind CAR, differences in fiber lengths, knob sequences, and other capsid proteins, such as penton bases, for different serotypes are consistent in certain adenoviruses. The efficiency in infecting the target cells can be determined. In this regard, it is interesting that Ad5 and Ad2 fibers can bind to fibronectin III and MHC class 1α2-derived peptides, and Ad3 fibers cannot. This suggests that adenoviruses can use cell receptors other than CAR (Hong et al., 1997). Serotypes 40 and 41 (F subgroup) are known to carry two fiber proteins with different shaft lengths. The long shaft 41L fiber is shown to bind CAR, while the short shaft 41S cannot bind CAR (Roelvink et al., 1998). The receptor for this short fiber is not known.
[0012]
Adenovirus gene delivery vector
Most adenoviral gene delivery vectors currently used in gene therapy are derived from serotype C adenovirus Ad2 or Ad5. These vectors have a deletion in the E1 region where new genetic information can be introduced. E1 deletion renders the recombinant virus replication defective. Recombinant adenovirus, particularly serotype 5, is a human xenograft in animal models and immunodeficient mice and has been widely demonstrated to be suitable for efficient in vivo gene transfer to liver, airway epithelium and solid tumors (Bout 1996, 1997; Blaese et al., 1995).
[0013]
Adenovirus-derived gene transfer vectors (adenovirus vectors) have several features that make them particularly useful for gene transfer:
1) The biology of adenovirus is well characterized
2) Adenovirus is not related to human serious pathology
3) This virus introduces its DNA into host cells very efficiently
4) This virus can infect a wide variety of cells and has a wide host range
5) This virus can be produced in large quantities with high titer
6) This virus can be made replication defective by deletion of the early region 1 (E1) of the viral genome (Brody and Crystal, 1994).
[0014]
However, there are still some disadvantages associated with the use of adenoviral vectors:
1) Adenoviruses, especially the serotypes Ad2 and Ad5 that have been studied in detail, usually elicit an immune response by the host into which they are introduced
2) It is not currently possible to target the virus to specific cells and tissues
3) Adenovirus replication and other functions are not always very suitable for cells seeking to provide additional genetic material.
4) Serotypes Ad2 or Ad5 are not ideally suited for delivering additional genetic material to organs other than the liver. The liver can be transduced particularly well with vectors derived from Ad2 or Ad5. Delivery of such vectors by blood flow leads to significant delivery of the vector to liver cells. In treatments where cell types other than hepatocytes need to be transduced, liver exclusion measures must be applied to prevent uptake of the vector by hepatocytes. Current methods rely on physical isolation of the vector from hepatocytes, most of which localize the vector and / or target organ by surgery, balloon angioplasty, or direct injection into the organ, for example by a needle. By being materialized. Liver exclusion is also performed by delivery of the vector to a compartment of the body that is essentially isolated from the blood stream, thereby preventing transport of the vector to the liver. Although these methods are mostly successful in avoiding significant delivery of the vector to the liver, most of these methods are crude, still leaky and / or have insufficient target tissue penetration . In some cases, inadvertent delivery of the vector to hepatocytes can be toxic to the patient. For example, the delivery of the herpes simplex virus (HSV) thymidine kinase (TK) gene to later kill mitotic cancer cells by administration of ganciclovir can also transduce significant amounts of hepatocytes by the vector. Is extremely dangerous. Significant delivery and subsequent expression of the HSV-TK gene into hepatocytes is associated with severe toxicity. Thus, there is a need for an individually safe vector that is given the property of reduced hepatocyte transduction efficiency.
[0015]
Brief description of the table
Table I: Oligonucleotides and degenerate oligonucleotides used to amplify DNA encoding fiber proteins derived from alternative adenovirus serotypes. (Bold letters represent NdeI restriction sites (AE), NsiI restriction sites (1-6, 8) or PacI restriction sites (7).)
[0016]
Table II: Biodistribution of chimeric adenovirus after intravenous tail vein injection. Each value represents luciferase activity / μg-total protein. Any value below 200 relative light units / μg-protein is considered background. ND = undecided
[0017]
Table III: CAR and integrin expression on the cell surface of endothelial and smooth muscle cells. 70%: Cells collected for FACS analysis at a cell density of 70% confluency. 100%: Cells collected for FACS analysis at a cell density of 100% confluency. PER.C6 cells were used as a control for antibody staining. The value represents the percentage of cells that express either one of CAR or integrin at a level above background. As a background control, HUVEC or HUVsmc was incubated with secondary rat anti-mouse IgG1-PE labeled antibody alone.
[0018]
Table IV: Measurement of transgene expression (luciferase activity) per μg of total cellular protein after infection of A549 cells.
[0019]
Summary of the Invention
  The present invention provides gene therapy methods, compounds and medicaments. The invention is particularly useful in gene therapy applications where endothelial cells and / or smooth muscle cells form a target cell type. The present invention relates to gene delivery provided with tissue tropism to at least endothelial cells and / or smooth muscle cells.VehicleAbout. Furthermore, the present invention provides gene delivery with tissue tropism removed from hepatocytes.VehicleAbout.
[0020]
Detailed Description of the Invention
It is an object of the present invention to provide materials and methods for overcoming the limitations of adenoviral vectors described above. In a broad sense, the present invention provides a new adenovirus derived from a serotype that is wholly or partly different from Ad5. Specific genes of serotypes with favorable characteristics can be combined into a single chimeric vector to yield a vector that is more suitable for a particular application. Preferred features include improved infectivity of specific target cells, reduced infectivity of unlabeled cells, improved virus stability, reduced uptake in antigen presenting cells (APC) or increased uptake on APC, target cells Reduced toxicity to humans, decreased neutralization in humans or animals, decreased or increased CTL responses in humans or animals, better and / or sustained transgene expression, increased ability to enter tissues, packaging cell lines However, this is not a limitation.
[0021]
One aspect of the invention facilitates the combination of the low immunogenicity of some adenoviruses with other adenoviral features that allow for efficient gene therapy. Such features include high specificity for certain host cells, good replication mechanisms for certain cells, high infection rates for certain host cells, low infection efficiency for non-target cells, high or low APC infection efficiency And so on. Thus, the present invention can provide a chimeric adenovirus having the useful properties of at least two adenoviruses having different serotypes.
[0022]
Typically, two or more of the non-exhaustive list are required to obtain adenoviruses that can efficiently introduce genetic material into host cells. Accordingly, the present invention provides an adenovirus-derived vector that can be used as a cassette for inserting different adenovirus genes from different adenovirus serotypes at the required site. In this way, a vector capable of generating a chimeric adenovirus can be obtained, and of course the gene of interest can also be inserted (eg, at the E1 site of the original adenovirus). In this way, the chimeric adeno Lewis produced can be adapted to the needs of certain hosts that require gene therapy for certain disorders. To enable production of this virus, packaging cells are generally required to produce a sufficient amount of safe chimeric adenovirus.
[0023]
  In one aspect, the present invention provides at least one fragment of an adenovirus fiber protein belonging to subgroup B.includingAn adenoviral vector is provided. The fiber protein may be a natural fiber protein of the adenoviral vector or may be derived from a serotype different from the serotype on which the adenoviral vector is based. In the latter case, the adenoviral vector of the present invention is a chimeric adenovirus that displays at least one fragment of a fiber protein derived from a subgroup B adenovirus, and that fragment comprises at least a fragment comprising a receptor binding sequence. Typically, such viruses will be produced using a vector (typically a plasmid, cosmid or baculovirus vector). Such vectors are also the subject of the present invention. Preferred vectors are those that can be used to generate chimeric recombinant viruses that are particularly suitable for the host to be treated and the disorder to be treated.
[0024]
  The present invention is a chimeric adenovirus based on adenovirus type 5 but having at least one fragment of a fiber sequence derived from adenovirus type 16, wherein the fiber fragment of Ad16 is involved in host cell binding. fragmentincludingAlso provide things. The present invention also relates to improved infection compared to adenoviruses belonging to other subgroups in certain host cells, such as, but not limited to, endothelial cells and smooth muscle cells of human or animal origin. Also provided are chimeric adenoviral vectors that exhibit sex. An important feature of the present invention is a means for producing chimeric viruses. Typically, it is not desirable to administer a batch of adenovirus containing replicating competent adenovirus to a host cell. Thus, in general, some genes (but at least one) are excluded from the adenoviral genome on the vector encoding the chimeric virus and are supplied to the genome of the cell that causes the vector to produce the chimeric adenovirus. It is desirable. Such cells are commonly referred to as packaging cells. Accordingly, the present invention also provides a packaging cell for producing the chimeric adenovirus of the present invention, which contains all elements necessary for the production of adenovirus and not present on the adenovirus vector of the present invention in trans. Typically, the vector and the packaging cell must be compatible with each other in that they have all the necessary elements but do not have duplicate elements that cause recombination competent viruses by recombination. Therefore, the present invention provides the packaging cell of the present invention and the recombinant vector of the present invention.includingSome provide kits of parts that are essentially free of sequence duplication between the cell and the vector leading to recombination resulting in the production of replication competent adenovirus. For certain applications, such as treatment aimed at eradication of tumor cells, the adenoviral vectors of the present invention are replication competent or within a specific cell type, such as tumor cells or tumor endothelial cells, It may be replicable under certain conditions.
[0025]
It is within the scope of the present invention to insert more genes or functional parts of those genes from the same or other serotypes into the adenoviral vector to replace the corresponding native sequence. Thus, for example, replacement of a fiber sequence with a corresponding sequence of another serotype, such as the serotype or other of another capsid gene such as a penton base or hexon. Can be combined with replacement of the different serotypes with corresponding sequences. Those skilled in the art will appreciate that other combinations not limited to the genes are contemplated and are within the scope of the present invention. The chimeric adenoviral vectors of the present invention can be derived from at least two different serotypes. This is because the vector has improved infection of target cells and / or reduced infection of non-target cells, improved virus stability, reduced immunogenicity in humans or animals (eg reduced uptake in APC, Preferred features such as reduced neutralization in the host and / or reduced cytotoxic T lymphocyte (CTL) response), increased tissue permeability, improved transgene expression persistence, and the like. In this regard, use capsid genes, such as penton and / or hexon genes, derived from low immunogenic serotypes defined by the absence or low amount of neutralizing antibodies in most hosts It is preferable. It is also preferred to use fiber and / or penton sequences derived from serotypes that show improved binding and internalization in target cells. Furthermore, it is preferred to delete from the viral vector the gene that results in the expression of the adenoviral gene in the target cell. In this respect, a vector from which all adenovirus genes have been deleted is also preferred. Furthermore, it is also preferable that the promoter driving the target gene to be expressed in the target cell is a cell type specific promoter.
[0026]
  In order to be able to precisely adapt the viral vector and freely give the desired properties to the chimeric virus, prepare a library of adenovirus genes and replace the gene to be exchanged with a plasmid or cosmid adenovirus. It is preferred to place the restriction site adjacent to the gene or sequence to be placed on the construct and to be exchanged. Preferred genes or sequences can be selected from the library and inserted into the adenovirus construct used to generate the virus. Typically, such methods involve several restriction and ligation steps and transfection of packaging cells.including. Adenoviral vectors are either partially transfected or transfected as two or more overlapping fragments, and the virus is generated by homologous recombination. For example, adenoviral vectors are assembled from two or more overlapping sequences for insertion or substitution of the gene of interest within the E1 region, insertion or substitution within the penton and / or hexon sequence, or insertion or substitution into the fiber sequence, for example. be able to. Accordingly, the present invention provides a method for producing a chimeric adenovirus having one or more desirable properties, such as a desired host range or reduced antigenicity, comprising one or more vectors of the present invention having a desired insertion site, At least a functional part of a fiber protein derived from an adenovirus serotype having a desired host range is inserted into the vector and / or a functional capsid protein derived from an adenovirus serotype having a relatively low antigenicity Inserting a portion and transfecting the vector into the packaging cells of the invention to produce chimeric viral particlesincludingProvide a method. Of course, other combinations of other viral genes from different serotypes can be inserted as disclosed herein above. Chimeric viruses having only one non-native sequence in addition to insertion or substitution of the gene of interest in the E1 region are also within the scope of the present invention.
[0027]
A typical immunogenic response to adenovirus is the production of neutralizing antibodies by the host. This is typically the reason for choosing less immunogenic serotype capsid proteins such as pentons, hexons and / or fibers.
[0028]
Of course, it may not be necessary to generate chimeric adenoviruses with complete proteins from different serotypes. It is well within the art to produce chimeric proteins, for example in the case of fiber proteins, it is clearly possible to have a shaft and knob from one serotype with one serotype base. In this way, the part of the protein responsible for the assembly of the viral particles can be derived from one serotype to promote the production of intact viral particles. Thus, the present invention also provides a chimeric adenovirus of the present invention, wherein the hexon, penton, fiber and / or other capsid proteins are chimeric proteins derived from different adenovirus serotypes. In addition to creating chimeric adenoviruses by exchanging whole or part of wild-type capsid (protein) genes etc., temperature stability, assembly, fixation, redirected infections, It is also within the scope of the present invention to insert non-adenovirus sequences that can be easily screened for favorable features such as altered immune responses and capsid (protein) genes carrying mutations such as point mutations, deletions, insertions, etc. included. Again, other chimeric combinations can be produced and are within the scope of the present invention.
[0029]
It has been demonstrated in mice and rats that systemic delivery of serotype recombinant adenoviruses commonly used for gene therapy in vivo captures more than 90% of the virus in the liver (Herz) 1993; Kass-Eisler et al., 1994; Huard et al., 1995). Human hepatocytes are also known to be efficiently transduced with adenovirus serotype 5 vectors (Castell, JV), Hernandez (D.), Gomez-Fox AM Foix, AM), Guillen, I., Donato, T. and Gomez-Lechon, MJ (1997) “Genez-Lechon, MJ” (1997) Adenoviral gene transfer into human hepatocytes: Analysis of the biochemical function of transduced cells (Adenovirus-mediated gene transfer into human hepatocytes: analysis of the biochemical functionality of transduced cells), Gene Ther. 4 (5), pages 455-464) . Thus, in vivo gene therapy by systemic delivery of Ad2 or Ad5-based vectors is significantly hampered by efficient virus uptake in the liver, resulting in undesirable virulence and fewer viruses available for target cell transduction. Become. Accordingly, it is a major concern of the present invention to alter the host cell range of adenovirus serotype 5 so that it can target other organs in vivo.
[0030]
Several methods have been studied or are still being studied to achieve re-directed infection of recombinant adenovirus serotype 5. Wickham et al., Α to Penton Base_vβ_ThreeAnd α_vβ_FiveThe RGD (Arg, Gly, Asp) motif in the penton base, which is thought to be responsible for integrin binding, was modified. They use this RGD motif as α_Fourβ_lSubstitution with another peptide motif specific for the receptor. This method allowed the adenovirus to be directed to specific target cells (Wickham et al., 1995). Krasnykh et al. (1998) used the HI loop available in the knob. This loop appears to be outside the knob trimer structure according to X-ray crystallography and therefore does not contribute to intramolecular interactions at the knob. Insertion of the FLAG coding sequence into the HI loop resulted in a fiber protein that could be trimerized, and it was further shown that viruses containing the FLAG sequence in the knob region could be made. Although the interaction between the FLAG-containing knob and CAR does not change, insertion of the ligand into the HI loop can lead to retargeting of the infection. Although successful cases of introducing changes to the adenovirus serotype 5 fiber and penton base have been reported, limited knowledge of the complex structure of the knob and the precise amino acid interaction with CAR is the only way to derive such a method. Making it cumbersome and difficult. The use of antibodies that bind CAR to specific cell receptors has also been described (Wickham et al., 1996; Rogers et al., 1997). However, this method is limited by the availability of specific antibodies and the complexity of gene therapy products.
[0031]
To overcome the above limitations, we used existing adenovirus fibers, penton base proteins, hexon proteins or other capsid proteins derived from other adenovirus serotypes. By creating a chimeric adenovirus serotype 5 library containing structural proteins of alternative adenovirus serotypes, we have developed a technology that allows for rapid screening of recombinant adenovirus vectors with favorable characteristics.
[0032]
  It is an object of the present invention to provide a method for making chimeric capsids that can target specific cell types in vitro and in vivo and thus have altered tropism for certain cell types. Protein or nucleic acid delivery of adenovirus or adenovirus capsid to specific cell types or tissuesVehicleIt is a further object of the present invention to provide methods and means that allow it to be used as
[0033]
The production of a chimeric adenovirus based on adenovirus serotype 5 with a modified late gene is described. To that end, three plasmids containing the complete adenovirus serotype 5 genome as a whole were constructed. From one of these plasmids, a portion of the DNA encoding the adenovirus serotype 5 fiber protein was removed and replaced with a linker DNA sequence that facilitates cloning. Next, this plasmid was used as a template for inserting a DNA encoding a fiber protein derived from a different adenovirus serotype. DNA sequences derived from different serotypes were obtained by using the polymerase chain reaction technique in combination with (degenerate) oligonucleotides. Any gene of interest can be cloned into the former E1 position in the genome of adenovirus serotype 5. A single transfection of all three plasmids forms a recombinant chimeric adenovirus. Alternatively, cloning of sequences obtained from a library of genes can be such that a chimeric adenoviral vector is constructed from one or two fragments. For example, one construct provides at least the sequences required for viral packaging and the left IRT, the expression cassette for the gene of interest, as well as all the sequences required for replication and virus formation that are not present in the packaging cell. Contains a sequence that overlaps with a second construct consisting of a non-native sequence. Thus, this new technique of libraries consisting of chimeric adenoviruses allows for rapid screening of improved recombinant adenoviral vectors for in vitro and in vivo gene therapy.
[0034]
The use of adenovirus type 5 for in vivo gene therapy is preferential to the inability to infect certain cell types such as human endothelial cells and smooth muscle cells, and infection of certain organs such as liver and spleen. Is limited by Specifically, this affects the treatment of cardiovascular diseases such as restenosis and pulmonary hypertension. Delivery of human ceNOS (constitutive endothelial nitric oxide synthase) by adenovirus has been proposed as a treatment for restenosis after percutaneous transluminal coronary angioplasty (PTCA). Restenosis is characterized by gradual arterial remodeling at the site of angioplasty, extracellular matrix formation and intimal hyperplasia (Schwartz et al., 1993; Carter et al., 1994; Shi) et al., 1996). NO is one of the vasoactive factors that has been shown to be lost after PTCA-induced endothelial barrier damage (Lloyd Jones and Bloch, 1996). Thus, restenosis can be prevented by restoring adenoviral levels using ceNOS after balloon-induced injury (Varenne et al., 1998). Non-limiting examples of other gene therapy applications in which the virus or chimeric virus of the present invention is superior to Ad2 or Ad5 viruses include production of proteins secreted in blood by endothelial cells, Treatment, prevention treatment of stenosis during vein transplantation, angiogenesis, heart failure, renal hypertension.
[0035]
In one aspect, the present invention describes adenoviral vectors that are particularly suitable for gene delivery to endothelial and smooth muscle cells, which are important for the treatment of cardiovascular disorders, among others. The present adenoviral vector preferably contains a functional portion derived from a B subgroup adenovirus or at least a fiber protein derived from an adenovirus belonging to the B subgroup, including at least the cell binding portion of the fiber protein. In a further preferred embodiment, the adenovirus vector is a chimeric vector based on adenovirus type 5, and contains at least a functional part of a fiber protein derived from adenovirus type 16.
[0036]
In another embodiment, the present invention provides an adenoviral vector or a chimeric adenoviral vector that escapes the liver after systemic administration. Preferably these adenoviral vectors are derived from the A, B, D or F subgroups, specifically serotypes 12, 16, 28 and 40 or contain at least the cell binding portion of the fiber protein derived from said adenovirus. To do.
[0037]
In any embodiment, the adenoviral vector can be derived from a serotype having the desired properties, or the adenoviral vector is based on an adenovirus belonging to one serotype and contains a sequence that includes the desired function of another serotype. It should be understood that these sequences replace the native sequence within the serotype.
[0038]
In another aspect, the present invention describes chimeric adenoviruses with altered tropisms different from adenovirus serotype 5 and methods for making these viruses. For example, a virus based on adenovirus serotype 5 but displaying any adenovirus fiber present in nature. This chimeric adenovirus serotype 5 can infect certain cell types with higher or lower efficiency in vitro and in vivo than adenovirus serotype 5. Such cells include endothelial cells, smooth muscle cells, dendritic cells, neuronal cells, glial cells, synovial cells, lung epithelial cells, hematopoietic stem cells, monocytes / macrophage cells, tumor cells, skeletal muscle cells, mesothelial cells There are synovial cells, but it is not limited to these.
[0039]
In another aspect, the present invention relates to the construction of a library comprising different portions of adenovirus serotype 5 in which one or more genes or sequences have been replaced with DNA from an alternative human or animal serotype and its Describe use. This set of constructs that encompass the entire adenovirus genome as a whole allows for the construction of unique chimeric adenoviruses specific to a particular disease, group of patients, or even an individual.
[0040]
In any aspect of the invention, the chimeric adenovirus may or may not contain a deletion in the E1 region and the insertion of a heterologous gene linked or not linked to a promoter. Furthermore, the chimeric adenovirus may or may not contain a deletion in the E3 region and the insertion of a heterologous gene linked to a promoter. Furthermore, the chimeric adenovirus may or may not contain a deletion in the E2 and / or E4 region and the insertion of a heterologous gene linked to a promoter. In the latter case, production of recombinant adenovirus requires E2 and / or E4 complementing cell lines. In fact, any gene in the genome of a viral vector can be removed and fed into trans. Thus, in extreme cases, chimeric viruses do not contain any adenoviral genes in their genome, and by definition are minimal adenoviral vectors. In this case, all adenoviral functions are supplied in trans using stable cell lines and / or transient expression of those genes. A method for producing a minimal adenoviral vector is described in WO97 / 00326, which is hereby incorporated by reference. In another example, an Ad / AAV chimeric molecule is packaged into an adenovirus capsid of the invention. A method for producing Ad / AAV chimeric vectors is described in EP97204085.1, which is hereby incorporated by reference. In principle, any nucleic acid can be provided with the adenovirus capsid of the invention.
[0041]
  In one aspect, the present invention provides gene delivery provided with tissue tropism at least to smooth muscle cells and / or endothelial cellsVehicleI will provide a. In another embodiment, the present invention provides gene delivery with at least tissue tropism removed from hepatocytes.VehicleI will provide a. Preferably, the gene deliveryVehicleIs provided with a tissue tropism for at least smooth muscle cells and / or endothelial cells and at least a tissue tropism for hepatocytes is removed. In a preferred embodiment of the present invention, the gene deliveryVehicleUses a fiber protein derived from subgroup B adenovirus, preferably adenovirus 16, to provide tissue tropism to at least smooth muscle cells and / or endothelial cells and / or tissue orientation to at least hepatocytes. Sex is removed. As a preferred aspect of the present invention, the gene deliveryVehicleContains the viral capsid. Preferably, the viral capsid comprises, in whole or in part, a viral capsid derived from a B subgroup adenovirus, preferably adenovirus 16, or a protein derived from a B subgroup adenovirus, preferably adenovirus 16 Or part of it. In a preferred embodiment of the invention, the viral capsid comprises a protein or fragment thereof derived from at least two different viruses, preferably adenovirus. In a preferred embodiment of this aspect of the invention, at least one of the viruses is a subgroup B adenovirus, preferably adenovirus 16.
[0042]
  In a preferred embodiment of the present invention, the gene deliveryVehicleContains adenovirus fiber protein or fragments thereof. The fiber protein is preferably derived from a subgroup B adenovirus, preferably adenovirus 16. The gene deliveryVehicleCan further include other fiber proteins or fragments thereof derived from other adenoviruses. The gene deliveryVehicleMay or may not contain other adenoviral proteins. The nucleic acid can be directly bound to the fiber protein or fragment thereof, but may be bound indirectly. Examples of indirect binding include the packaging of nucleic acids into adenovirus capsids or the packaging of nucleic acids into liposomes, where the fiber protein or fragment thereof is incorporated into or bound to the adenovirus capsid. It is not limited. Direct binding of a nucleic acid to a fiber protein or fragment thereof is when the fiber protein or fragment thereof is not part of a complex or when the fiber protein or fragment thereof is part of a complex such as an adenovirus capsid Can be done.
[0043]
  As one aspect of the present invention, gene delivery comprising adenovirus fiber proteinVehicleProvided that the fiber protein comprises a tissue-determining fragment of subgroup B adenovirus, preferably adenovirus 16. The adenovirus fiber protein contains three functional domains. One domain base is responsible for anchoring the fiber to the penton base of the adenovirus capsid. Another domain knob is responsible for receptor recognition, while the shaft domain functions as a spacer separating the base from the knob. Various domains may have other functions. For example, the shaft is probably also involved in target cell specificity. Each of the aforementioned domains can be used to define fiber fragments. However, fragments can be identified in other ways. For example, the knob domain is a receptor binding fragment and a shaft binding fragment.including. The base domain is a penton base binding fragment and a shaft binding fragmentincluding. In addition, the shaft is a stretch of repetitive amino acidsincluding. Each of these repeated stretches can be a fragment.
[0044]
  The tissue tropism determining fragment of a fiber protein can be a single fragment of fiber protein or a combination of at least one fragment of fiber protein, wherein the tissue tropism determining fragment is alone or In combination with the viral capsid, preferably positive, but not necessarily, gene deliveryVehicleDetermines the efficiency with which can transduce a given cell or cell type. Tissue tropism for hepatocytes means tissue tropism for cells present in the liver, preferably hepatocytes.
[0045]
Tissue tropism for a specific tissue can be provided by increasing the efficiency with which cells of the tissue are transduced, or tissue tropism for a specific tissue is transduced by cells other than cells of the tissue. Can be provided by reducing the efficiency.
[0046]
Fiber protein has tissue tropism determinism. The most detailed fragment of the fiber protein involved in tissue tropism is the knob domain. However, the shaft domain of fiber protein also has tissue tropism determinism. However, not all of the tissue tropism determinants of adenovirus capsids are incorporated into fiber proteins.
[0047]
In a preferred embodiment of the invention, a fiber protein derived from a subgroup B adenovirus, preferably adenovirus 16, is combined with a non-fiber capsid protein derived from a subgroup C adenovirus, preferably adenovirus 5.
[0048]
  As one aspect of the present invention, gene delivery comprising a nucleic acid derived from an adenovirusVehicleIs provided. In a preferred embodiment of the invention, the adenovirus nucleic acid comprises at least one nucleic acid sequence encoding a fiber protein comprising at least a subgroup B adenovirus fiber protein, preferably a tissue-tropic determining fragment of adenovirus 16. In a preferred aspect, the adenovirus comprises nucleic acids derived from at least two different adenoviruses. In a preferred aspect, the adenovirus comprises nucleic acids derived from at least two different adenoviruses, and the at least one nucleic acid sequence is a tissue tropism determining fragment of at least a B subgroup adenovirus fiber protein, preferably adenovirus 16. Which encodes a fiber protein containing
[0049]
In a preferred embodiment of the invention, the adenoviral nucleic acid is modified such that the replication capacity of the adenoviral nucleic acid in target cells is diminished or disabled. This can be achieved by inactivating or deleting the gene encoding the early region 1 protein.
[0050]
  In another preferred embodiment, the adenoviral nucleic acid is modified such that the host immune system's ability to initiate an immune response against the adenoviral protein encoded by the adenoviral nucleic acid is weakened or disabled. . This can be achieved by deletion of the gene encoding the protein of early region 2 and / or early region 4. Alternatively, if the gene encoding the early region 3 protein is deleted or, conversely, considering the anti-immune system function of a part of the protein encoded by the gene in the early region 3, the expression of the early region 3 protein is It can be strengthened for several purposes. In addition, the present adenovirus nucleic acid can be modified by a combination of two or more of the specific modifications of the adenovirus nucleic acid described above. When an essential gene is deleted from adenovirus nucleic acid, adenovirus nucleic acid, adenovirus vector,VehicleOr it is clear that these genes must be complemented in the cell in which the chimeric capsid is to be produced. In addition, adenoviral nucleic acid can be obtained by a method other than that described above, for example, a capsid protein or a fragment thereof derived from another serotype that humans do not have neutralizing antibodies against, or have low levels. By exchanging with the fragments, modifications can also be made to weaken or disable the host immune system's ability to initiate an immune response against the adenoviral protein encoded by the adenoviral nucleic acid. Another example of this is the exchange of genes encoding capsid proteins with genes encoding capsid proteins from other serotypes. The capsid protein or fragment thereof can also be exchanged for other capsid proteins or fragments thereof where each individual is unable to generate an immune response against it or has a low capacity.
[0051]
Adenoviral nucleic acid is further or alternatively modified by inactivating or deleting genes encoding adenoviral late proteins such as hexon, penton, fiber and / or protein IX instead of one or more of the modifications described above. Can, but is not limited to.
[0052]
In a preferred embodiment of the present invention, all genes encoding adenoviral proteins are deleted from the adenoviral nucleic acid, making the nucleic acid a minimal adenoviral vector.
[0053]
In another preferred embodiment of the present invention, the adenoviral nucleic acid is an Ad / AAV chimeric vector in which at least adeno-associated virus (AAV) integration means is integrated into the adenoviral nucleic acid.
[0054]
In a preferred embodiment of the present invention, the vector or nucleic acid of the present invention, which may or may not be identical, further comprises at least one non-adenoviral gene. Preferably, at least one of the non-adenovirus genes is apolipoprotein, ceNOS, herpes simplex virus thymidine kinase, interleukin-3, interleukin-1α, angiostatin and other (anti) angiogenic proteins, antiproliferative proteins, Encodes vascular endothelial growth factor (VGAF), basic fibroblast growth factor (bFGF), hypoxia-inducible factor 1α (HIF-1α), PAI-1 or smooth muscle cell anti-migration protein Selected from the group of genes.
[0055]
  In another aspect, the present invention relates to gene delivery provided with tissue tropism to at least smooth muscle cells and / or endothelial cells.VehicleProvide cells for producing. In another aspect, the present invention provides gene delivery with at least tissue tropism removed from hepatocytesVehicleProvide cells for producing. In another aspect, the present invention provides gene delivery in which tissue tropism is given to at least smooth muscle cells and / or endothelial cells, and tissue tropism is removed to at least hepatocytesVehicleA cell for producing is provided. In a preferred embodiment of the invention, the cell is an adenovirus packaging cell where adenovirus nucleic acid is packaged into an adenovirus capsid. As one aspect of the adenovirus packaging cell of the present invention, all proteins necessary for the replication and packaging of adenovirus nucleic acid are incorporated into the adenovirus nucleic acid except for the protein encoded by the initial region 1. Provided by. In this aspect of the invention, the protein encoded by early region 1 can be encoded by a gene integrated into the cellular genomic DNA. In a preferred embodiment of the invention, the cell is PER.C6 (ECACC accession number 96022940). In general, if the gene product required for replication of the adenoviral nucleic acid and packaging into the adenoviral capsid is not provided by the adenoviral nucleic acid, they can be obtained by transient transfection of the packaging cell or by stable transformation. Provided by packaging cells. However, the gene product provided by the packaging cell can also be provided by a gene present on the adenoviral nucleic acid. For example, fiber proteins can be provided by packaging cells, for example via transient transfection, or can be encoded by adenoviral nucleic acids. This feature is notably the fiber protein from two different virusesincludingCan be used to make adenovirus capsids.
[0056]
  Gene delivery of the present inventionVehicleAre useful for the treatment of cardiovascular diseases or diseases that can be treated by nucleic acid delivery to endothelial cells or smooth muscle cells. Non-limiting examples of the latter include, for example, cancer, where the transferred nucleic acid encodes an anti-angiogenic protein.
[0057]
  Gene delivery of the present inventionVehicleCan be used as a drug for the treatment of the aforementioned diseases. Alternatively, the gene delivery of the present inventionVehicleCan be used for the manufacture of a medicament for the treatment of the aforementioned diseases.
[0058]
  In one aspect, the present invention provides an adenoviral capsid with or given tissue tropism for smooth muscle cells and / or endothelial cells, wherein the capsid is preferably a protein derived from at least two different adenovirusesIncluding, At least the tissue tropism-determining fragment of the fiber protein is provided from subgroup B adenovirus, preferably adenovirus 16. In another aspect, the present invention relates to an adenovirus capsid from which tissue tropism to hepatocytes has been removed, wherein the capsid is preferably a protein derived from at least two different adenoviruses.IncludingAt least the tissue-tropic determining fragment of the fiber protein is derived from a subgroup B adenovirus—preferably adenovirus 16.
[0059]
In one aspect, the invention consists of the use of an adenoviral capsid to deliver nucleic acids to smooth muscle cells and / or endothelial cells. In another embodiment, the present invention consists of the use of an adenovirus capsid to inhibit delivery of nucleic acids to hepatocytes. The adenoviral capsids of the invention can be used to treat cardiovascular diseases or diseases that can be treated by nucleic acid delivery to endothelial cells or smooth muscle cells. An example of the latter is cancer, for example, where the transferred nucleic acid contains a gene encoding an anti-angiogenic protein.
[0060]
The adenovirus capsid of the present invention can be used as a drug for treating the aforementioned diseases. Alternatively, the adenovirus capsid of the present invention can be used for the manufacture of a medicament for the treatment of the disease.
[0061]
In another aspect of the invention there is provided a construct pBr / Ad.BamRΔFib comprising the adenovirus 5 sequences 21562-31094 and 32794-35938.
[0062]
In another aspect of the invention, there is provided a construct pBr / AdBamRfib16 comprising the adenovirus 5 sequences 21562-31094 and 32794-35938 and further comprising an adenovirus 16 gene encoding a fiber protein.
[0063]
In yet another aspect of the invention, the adenovirus 5 sequences 21562-31094 and 32794-35938 are included, the adenovirus 16 gene encoding the fiber protein is further included, and the adenovirus in the non-adenovirus sequence backbone of the construct A construct pBr / AdBamR.pac / fib16 is provided that contains a unique PacI site near the 5 right terminal repeat.
[0064]
In another aspect of the invention, there is provided a construct pWE / Ad.AflIIrITRfib16 comprising Ad5 sequences 3534-31094 and 32794-35938 and further comprising an adenovirus 16 gene encoding a fiber protein.
[0065]
In another aspect of the invention, there is provided a construct pWE / Ad.AflIIrITRDE2Afib16 comprising Ad5 sequences 35342-22443 and 24033-31094 and 32794-35938 and further comprising an adenovirus 16 gene encoding a fiber protein.
[0066]
In the sequence numbering, numbers are represented so far, but not thereafter.
[0067]
  In a preferred embodiment of the present invention, the construct provides gene delivery with tissue tropism to smooth muscle cells and / or endothelial cells.VehicleOr used to make adenovirus capsids.
[0068]
  In another aspect, the present invention provides an adenoviral vector comprising a number of selected non-adenoviral nucleic acids or gene delivery that may or may not be identicalVehicleProvide the library. In another aspect of the invention, the adenoviral gene encoding the capsid protein is a protein derived from at least two different adenovirusesincludingUsed to generate a library of adenovirus capsids, which are preferably derived from two different serotypes, preferably one serotype is a subgroup B adenovirus. In a particularly preferred embodiment of the invention, a library of adenovirus capsids comprising proteins from at least two different adenoviruses, wherein at least the tissue-tropic determining fragment of the fiber protein is a subgroup B adenovirus-preferably an adenovirus. Those derived from virus 16 are produced.
[0069]
  The fiber protein of adenovirus 16 is preferably the sequence described in FIG.including. However, within the scope of the present invention, similar sequences can be obtained by using codon degeneracy. Alternatively, amino acid substitutions or insertions or deletions may be made unless the tissue tropism is significantly altered. Such amino acid substitutions can be made within or outside the same polar group.
[0070]
In the following, the present invention is illustrated by several non-limiting examples.
[0071]
Example
Example 1: Adenovirus serotype with chimeric fiber protein Five Production of viruses
Code the fiber DNA Of adenovirus template clone lacking
The fiber coding sequence of adenovirus serotype 5 is located between nucleotides 31042 and 32787. In order to remove the adenovirus serotype 5 DNA encoding the fiber, we started with the construct pBr / Ad.Bam-rITR (FIG. 1; ECACC accession number P97082122). From this construct, one NdeI site was first removed. For this purpose, pBr322 plasmid DNA was digested with NdeI, and the overhanging end was supplemented with Klenow enzyme. The pBr322 plasmid was then religated, digested with NdeI, and transformed into E. coli DH5α. The resulting pBr / ΔNdeI plasmid was digested with ScaI and SalI, and the resulting 3198 bp vector fragment was ligated to the 15349 bp ScaI-SalI fragment derived from pBr / Ad.BamrITR, thereby containing a unique NdeI site. pBr / Ad.Bam-rITRΔNdeI was obtained. Next, PCR was performed using oligonucleotides “NY-up” and “NY-down” (FIG. 2). To facilitate cloning of amplified fiber DNA, both NdeI and NsiI restriction sites were introduced during amplification. Amplification consisted of 25 cycles of 94 ° C for 45 seconds, 60 ° C for 1 minute and 72 ° C for 45 seconds, respectively. This PCR reaction consists of 25 pmol of oligonucleotide NY-up or NY-down, 2 mM dNTP, 1.5 mM MgCl₂PCR buffer containing 1 unit of Elongase thermostable polymerase (Gibco, The Netherlands). When one-tenth of the PCR product was run on an agarose gel, it was revealed that the expected DNA fragment of ± 2200 bp was amplified. The PCR fragment was then purified using the Geneclean kit system (Bio101). Next, both the construct pBr / Ad.Bam-rITRΔNdeI and the PCR product were digested with restriction enzymes NdeI and SbfI. Next, pBr / Ad.BamRΔFib (FIG. 3) was prepared by cloning the PCR fragment into NdeI and SbfI sites using T4 ligase enzyme.
[0072]
Amplification of fiber sequences from adenovirus serotypes.
Degenerate oligonucleotides were synthesized to allow amplification of DNA encoding fiber proteins from alternative serotypes. To that end, we first aligned known DNA sequences encoding alternative serotype fiber proteins to identify regions conserved in both the tail and knob regions of the fiber protein. (Degenerate) oligonucleotides were synthesized from their alignments containing nucleotide sequences of 19 serotypes representing all 6 subgroups (see Table I). Table 3 also shows combinations of oligonucleotides used to amplify DNA encoding a specific serotype fiber protein. Amplification reaction (50 μl) is 2 mM dNTP, 25 pmol of each oligonucleotide, standard 1 × PCR buffer, 1.5 mM MgCl per reaction.₂And 1 unit of Pwo thermostable polymerase (Boehringer Mannheim). The cycler program included 20 cycles of 94 seconds at 30 seconds, 60-64 degrees Celsius for 60 seconds, and 72 degrees Celsius for 120 seconds. One-tenth of the PCR product was run on an agarose gel to prove that the DNA fragment was amplified. Two independent PCR reactions were performed with each different template.
[0073]
Chimeric adenovirus DNA Building construction
All amplified fiber DNA and vectors (pBr / Ad.BamRΔFib) were digested with NdeI and NsiI. The digested DNA was then run on an agarose gel and each fragment was isolated from the gel and purified using a Geneclean kit (Bio101). Then, these PCR fragments were cloned into the NdeI and NsiI sites of pBr / AdBamRΔFib to prepare pBr / AdBamRFibXX (where XX represents the serotype number from which the fiber DNA was isolated). In order to confirm correct amplification, the insert generated by PCR was sequenced. The resulting sequences for various fiber genes are shown in FIG.
[0074]
Production of chimeric recombinant adenovirus with fiber protein
In order to allow efficient production of chimeric viruses, the AvrII fragment of the pBr / AdBamRFib16, pBr / AdBamRFib28, pBr / AdBamRFib40-L construct was transformed into the vector pBr / Ad.Bam-rITR.pac # 8 (ECACC accession number # P97082121). ) To replace the corresponding sequence in this vector. pBr / Ad.Bam-rITR.pac # 8 has the same adenoviral insert as pBr / Ad.Bam-rITR, but has a PacI site near its rITR, which separates the ITR from the vector sequence. Enable. The construct pWE / Ad.AflII-Eco was made as follows. pWE.pac was digested with ClaI and its 5 'protruding end was supplemented with Klenow enzyme. The DNA was then digested with PacI and isolated from an agarose gel. pWE / AflIIrITR was digested with EcoRI, treated with Klenow enzyme, and then digested with PacI. A large 24 kb fragment containing the adenovirus sequence was isolated from an agarose gel and ligated into the CWE-digested blunt-ended pWE.Pac vector. Clontech's ligation express kit was used. After transformation of Stratagene XL10-gold cells, clones containing the expected construct were identified. pWE / Ad.AlfII-Eco contains the Ad5 sequence of base pairs 3534-27336. Adenovirus production of three constructs, pClipsal-Luc digested with SalI (FIG. 5), pWE / Ad.AflII-Eco digested with PacI and EcoRI, and pBr / AdBamR.pac / fibXX digested with BamHI and PacI Cells (PER.C6, Fallaux et al., 1998) were transfected. FIG. 6 is a schematic diagram of the methods and fragments used for the production of chimeric viruses. Since only pBr / Ad.BamRfib12 was used without subcloning into a PacI-containing vector, PacI was not released from the vector sequence, but was digested with ClaI, which releases an approximately 160 bp vector sequence bound to the right ITR. Furthermore, since pBr / Ad.BamRfib12 and pBr / Ad.BamRfib28 contain an internal BamHI site in their fiber sequence, they were digested with SalI which cuts in the vector sequence adjacent to the BamHI site. For transfection, 2 μg of pCLIPsal-Luc and 4 μg of both pWE / Ad.AflII-Eco and pBr / AdBamR.pac / fibXX were diluted in serum-free DMEM to a total volume of 100 μl. To this DNA suspension, 100 μl of 2.5-fold diluted lipofectamine (Gibco) in serum-free medium was added. After 30 minutes at room temperature, the DNA-lipofectamine complex solution is added to 2.5 ml of serum-free DMEM, which is then added to T25 cm.²Added to tissue culture flask. This flask is 1 x 10 24 hours before transfection.⁶PER.C6 cells seeded at cell / flask density were included. Two hours later, the medium containing the DNA-lipofectamine complex was diluted once by adding 2.5 mL of DMEM supplemented with 20% fetal calf serum. After another 24 hours, the medium was replaced with fresh DMEM supplemented with 10% fetal calf serum. Cells were cultured for 6-8 days, then harvested and thawed / frozen 3 times. Cell debris was removed by centrifugation at 3000 rpm for 5 minutes at room temperature. Using 3 to 5 ml of the supernatant (12.5 ml), PER.C6 cells (T80cm²The tissue culture flask) was again infected. This reinfection results in a complete cytopathic effect (CPE) after 5-6 days, after which the adenovirus is collected as described above.
[0075]
Production of fiber chimeric adenovirus
1 to 1.5 × 10 10 of the above crude lysate suspension-cultured.⁶ A 1 liter fermentor containing PER.C6 cells / ml was inoculated. Three days after inoculation, cells were harvested and pelleted by centrifuging at 1750 rpm for 10 minutes at room temperature. Next, the chimeric adenovirus present in the pelleted cells was extracted and purified by the following post-treatment method. 50 ml of 10 mM NaPO pellet_Four ^-And frozen at -20 ° C. After melting at 37 ° C., 5.6 ml of deoxycholate (5% w / v) was added and then the solution was homogenized. The solution was then incubated at 37 ° C. for 15 minutes in order to completely destroy the cells. After homogenizing the solution, 1875 μl of (1M) MgCl₂ ^-And 5 ml of 100% glycerol was added. After adding 375 μl DNase (10 mg / ml), the solution was incubated at 37 ° C. for 30 minutes. Cell debris was removed by centrifugation at 1880 × g for 30 minutes at room temperature without braking. The supernatant was then purified from the protein by subjecting it to 10 ml freon. Centrifuge at 2000 rpm for 15 minutes at room temperature without applying the brakes, you can see three bands, of which the upper band corresponds to adenovirus. This band was isolated by pipetting and then applied to a Tris / HCl (1M) buffered cesium chloride block gradient (range: 1.2 to 1.4 g / ml). Since the virus did not migrate into the 1.4 g / ml cesium chloride solution in contrast to the other components, the virus was purified from residual proteins and cell debris after centrifugation at 10 ° C. and 21000 rpm for 2.5 hours. After isolation of the virus band, a second purification using a continuous gradient of 1.33 g / ml cesium chloride buffered with Tris / HCl (1 M) is performed. After placing the virus on top of this gradient, the virus is centrifuged at 10 ° C. and 55000 rpm for 17 hours. Next, the virus band is separated, 30 μl of sucrose (50 w / v) is added, and then excess cesium chloride is removed by three dialysis each consisting of 1 hour. For dialysis, the virus is transferred to a dialysis slide (Slide-a-lizer, cutoff 10000 kDa, Pierce, USA). The buffer used for dialysis was sucrose of increasing concentration (from 1st to 3rd: 30ml, 60ml and 150ml sucrose (50% (w / v) /1.5 liter PBS, all 7.5ml 2% (w / v) CaMgCl₂PBS supplemented. After dialysis, the virus is removed from the slide-a-lizer and then divided into 25 and 100 μl aliquots, after which the virus is stored at −85 ° C. To determine the number of virus particles per ml, 50 μl of the virus batch is subjected to high pressure liquid chromatography (HPLC) as described by Shamram et al. (1997). The virus titer was found to be in the same range as the Ad5.Luc virus batch (Ad5.Luc: 2.2 × 10¹¹vp / ml; Ad5.LucFib12: 1.3 × 10¹¹vp / ml; Ad5.LucFib16: 3.1 × 10¹²vp / ml; Ad5.LucFib28: 5.4 × 10^Tenvp / ml Ad5.LucFib40-L: 1.6 × 10¹²vp / ml).
[0076]
Example 2: Biodistribution of chimeric virus after intravenous and tail vein injection in rats
To examine the biodistribution of chimeric adenovirus carrying fiber 12, 16, 28 or 40-2, each virus batch made 1 x 10^TenAfter diluting the particles in 1 ml PBS, the virus was injected into the tail vein of adult male Wag / Rij rats (3 animals / virus). As a control, Ad5 carrying the luciferase transgene was used. Forty-eight hours after virus administration, rats were sacrificed and the liver, spleen, lung, kidney, heart and brain were dissected. These organs were then mixed with 1 ml of lysis buffer (1% Triton X-100 / PBS) and chopped for 30 seconds to obtain a protein lysate. The protein lysate was then tested for the presence of transgene expression (luciferase activity) and the protein concentration determined to represent luciferase activity per μg of protein. The results shown in Table II reveal that no fiber chimera is specifically targeted to the liver or spleen, in contrast to the adenovirus serotype 5 control. This experiment shows that adenovirus uptake by the liver can be avoided by utilizing fibers of other serotypes. It also indicates that liver uptake does not correlate with fiber shaft length or is determined only by the fiber knob's ability to bind to CAR. The fibers used have different shaft lengths, with the exception of fiber 16, except for subgroups known to have fibers capable of binding CAR (Roelvink et al., 1998).
[0077]
Example 3: Chimeric virus shows differences in endothelial and smooth muscle cell transduction
A) Infection of human endothelial cells
Human endothelial cells (HUVEC) were isolated, cultured and characterized as previously described (Jaffe et al., 1973; Wijnberg et al., 1997). Briefly, 20 mM HEPES, pH 7.3 (Flow Labs. Irvine, Scotland), 10% (v / v) human serum (local blood bank), 10% (v / v) fever Inactivated bovine newborn serum (NBCS) (GIBCO BRL, Gaithersburg, Md.), 150 μg / ml crude endothelial growth factor, 5 U / ml heparin (Leo Pharmaceuticals (Leo) In M119 supplemented with Pharmaceutics Products), Netherlands Weesp), penicillin (100 IU / ml) / streptomycin (100 μg / ml) (Boehringer Mannheim, Mannheim, Germany) in gelatin-coated culture dishes Cells at 37 ° C, 5% (v / v) CO₂The cells were cultured in / 95% (v / v) air. Cells used for the experiment were passage numbers 1-3. In the first set of experiments, 40,000 HUVEC cells (pool from 4 individuals) were seeded in each well of a 24-well plate in a total volume of 200 μl. After 24 hours of seeding, the cells were washed with PBS, and 200 μl of DMEM supplemented with 2% FCS was added to the cells. This medium contained various amounts of virus (MOI = 50, 250, 1000, 2500, 5000 and 10000). The viruses used were fiber chimeras 12, 16, 28 and 40-L in addition to the control Ad5 (3 each infection). Two hours after virus addition, the medium was replaced with normal medium. After an additional 48 hours, cells were washed and lysed by the addition of 100 μl lysis buffer. FIG. 7a shows the results for transgene expression per microgram of total protein after infection of HUVEC cells. These results show that fiber chimeras 12 and 28 cannot infect HUVEC cells, 40-L infects HUVEC with similar efficiency as the control Ad5 virus, and fiber chimera 16 infects HUVEC significantly better. Is shown. In the next set of experiments (n = 8), HUVEC compared the fiber 16 chimera with the Ad5.Luc vector for luciferase activity after transduction with each virus at 2500 viral particles per cell. These experiments demonstrated that fiber 16 gave an average 8.1-fold higher luciferase activity (SD ± 4.6) compared to Ad5. In the next experiment, an equal number of virus particles was added to the wells of a 24-well plate containing different HUVEC cell concentrations. This experiment was performed because HUVEC is known to reduce the efficiency of its infection by adenovirus serotype 5 when those cells reach confluency. To that end, HUVECs were seeded at a density of 22500, 45000, 90000 and 135000 cells per well of a 24-well plate (3 in each). After 24 hours, the cells are 4.5 x 10 as described above.⁸Infected with medium containing virus particles. The virus used was chimeric fiber 16 in addition to control adenovirus serotype 5. Transgene expression (RLU) results per microgram of protein determined 48 hours after infection (see Figure 7b) show that even when HUVEC cells are 100% confluent, which better mimics the in vivo situation. 16 shows that the chimeric adenovirus is more suitable for infecting HUVEC cells. Since the luciferase marker gene does not give information on the number of cells infected, another experiment was performed using adenovirus serotype 5 and fiber 16 chimeras carrying green fluorescent protein (GFP) as the marker gene. This protein expression can be detected with a flow cytometer that provides information on the percentage of cells transduced and the fluorescence per cell. In this experiment, cells were seeded at a concentration of 40,000 cells per well and exposed to the virus for 2 hours. The virus used was Ad5.GFP (8.4 x 10¹¹vp / ml) and Ad5.Fib16.GFP (5.1 × 10¹¹vp / ml). Cells were exposed to a virus concentration of 500 virus particles per cell. 48 hours after virus exposure, fiber 16 virus has a higher transgene per cell because the median fluorescence value, a parameter indicating GFP expression per cell, is higher for fiber 16 compared to Ad5. Giving expression levels was verified by analysis by flow cytometry (FIG. 7c). Thus, these results are consistent and demonstrate that Fiber 16 chimeric virus is better suited to infect human primary endothelial cells compared to Ad5.
[0078]
B) Human smooth muscle cell infection
Smooth muscle cells were isolated after EC isolation (Weinberg et al., 1997). Veins were incubated with medium (DMEM supplemented with penicillin / streptomycin) containing 0.075% (w / v) collagenase (Worthington Biochemical, Freehold, NJ, USA). After 45 minutes, the culture medium containing detached cells was washed away from the vein. Cells are washed and 37 ° C, 5% (v / v) CO₂The cells were cultured in a gelatin-coated dish in a culture medium supplemented with 10% fetal bovine serum and 10% human serum in a / 95% (v / v) atmosphere. Cells used for the experiments were passage numbers 3-6. We first tested a panel of chimeric fiber virus and control adenovirus serotype 5 for infection of human smooth muscle cells. To that end, 40,000 human umbilical vein smooth muscle cells (HUVsmc) were seeded in wells of 24-well plates in a total volume of 200 μl. Twenty-four hours after seeding, the cells were washed with PBS and then 200 μl of DMEM supplemented with 2% FCS was added to the cells. This medium contained various amounts of virus (MOI = 50, 250, 1250, 2500 and 5000). The viruses used were fiber chimeras 12, 16, 28 and 40-L in addition to the control Ad5 (3 each infection). Two hours after virus addition, the medium was replaced with normal medium. After an additional 48 hours, cells were washed and lysed by adding 100 μl lysis buffer. FIG. 8a shows the results of transgene expression per microgram of total protein after infection with HUVsmc cells. These results show that fiber chimeras 12 and 28 cannot infect HUVsmc cells, 40-L infects HUVsmc with the same efficiency as the control Ad5 virus, and fiber chimera 16 infects HUVsmc significantly better. Show. In the next set of experiments, saphenous vein, iliac artery, left internal mammary artery (LIMA) and aortic smooth muscle cells were infected with fiber 16 chimera and Ad5 (both carrying luciferase as a marker gene). Examined. These experiments (n = 11) revealed that fiber 16 chimeras gave an average 61.4-fold higher level of luciferase activity (SD ± 54.8) compared to Ad5. A high standard deviation (SD) is obtained due to the finding that the adenovirus used has different efficiencies for infection of SMCs derived from different human blood vessels. In the next experiment, an equal number of virus particles was added to the wells of a 24-well plate containing different HUVsmc cell concentration confluency. To that end, HUVsmc were seeded at a density of 10000, 20000, 40000, 60000 and 80000 cells per well of a 24-well plate (3 in each). After 24 hours, the cells are 2 × 10 2 as before.⁸Infected with medium containing virus particles. The virus used was chimeric fiber 16 in addition to control adenovirus serotype 5. Transgene expression (RLU) results per microgram of protein (RLU) determined 48 hours after infection (see FIG. 8b) show that even when smooth muscle cells are 100% confluent that better mimics the in vivo situation. Fiber 16 chimeric adenovirus is shown to be more suitable for infecting smooth muscle cells. To identify the number of SMCs transduced with fiber 16 chimera and Ad5, we conducted a transduction experiment with Ad5.GFP and Ad5Fib16.GFP (the same batch used for EC infection). Human umbilical vein SMCs were seeded in 24-well plates at a concentration of 60000 cells per well and exposed to 500 or 5000 viral particles of Ad5.GFP or Ad5Fib16.GFP per cell for 2 hours. Cells were harvested 48 hours after exposure and analyzed using a flow cytometer. Since the GFP expression measured after transduction of 5000 virus particles with Ad5.GFP is equal to the GFP expression after transduction with a fiber 16 chimera of 500 virus particles per cell (FIG. 8c), the results obtained are fiber 16 We show that the mutant gives about 10 times higher transduction of SMC.
[0079]
C) B subgroup fiber mutants other than fiber 16
The shaft and knob of the fiber 16 are derived from adenovirus serotype 16, and as described above, based on the hemagglutination test, DNA restriction pattern and neutralization test belonging to the B subgroup, the B subgroup virus is further classified into the B1 subgroup. It is subdivided into B2 subgroups (Wadell et al., 1984). Members of the B1 subgroup include serotypes 3, 7, 16, 21, and 51. Members of the B2 subgroup include 11, 14, 34 and 35. To investigate whether the increased infection of smooth muscle cells is a trade of all fibers from the B subgroup or is unique to one or more B subgroup fiber molecules, we And fiber 51 (both B1 subgroup) were compared to fiber 11 and fiber 35 (both B2 subgroup). To that end, HUVsmc was infected with increasing amounts of virus particles per cell (156, 312, 625, 1250, 2500, 5000). All fiber mutants carry the luciferase marker gene (Ad5Fib11.Luc: 1.1 × 10¹²vp / ml; Ad5Fib35Luc: 1.4 × 10¹²vp / ml; Ad5Fib51Luc: 1.0 × 10¹²vp / ml). According to the measured luciferase activity shown in FIG. 8d, efficient infection of SMC is not a common trade for all B subgroup fiber molecules. Obviously, fiber 16 and fiber 11 are more suitable for SMC infection than fiber 35 and fiber 51. Nevertheless, all B subgroup fiber mutants tested were better infected with SMC compared to Ad5.
[0080]
D) Organ culture experiment
Next, the inventors confirmed whether the differences observed in EC and SMC transduction using fiber 16 chimera or Ad5 could be demonstrated in organ culture experiments. In this regard, we focused on the following tissues: 1) Human saphenous vein: vein used in about 80% of all clinical vein grafts
2) Human pericardium / epicardium: for delivery of recombinant adenovirus to pericardial fluid producing the protein of interest from a transgene carried by adenovirus after infection of pericardium or epicardial cells
3) Human coronary artery: For percutaneous transluminal coronary angioplasty (PTCA) to prevent restenosis. Of the coronary arteries, we have left artery descending (LAD) and right coronary artery (RCA).
Focused on.
[0081]
A portion of the human saphenous vein after vein transplantation was sliced into approximately 0.5 cm sections. Next, these sections (n = 3) are 5 x 10 per ml.^TenIncubated in 200 ml of virus particles for 2 hours. After 2 hours of virus exposure, the sections were washed with PBS and 10% CO2.₂The cells were further cultured for 48 hours at 37 ° C in an incubator. The sections were then washed, fixed and stained for LacZ transgene expression. The virus is Ad5.LacZ (2.2 × 10¹²vp / ml), fiber 16 chimera Ad5Fib16.LacZ (5.2 × 10¹¹vp / ml) and fiber 51 chimeric Ad5Fib51.LacZ (2.1 x 10)¹²vp / ml). A section of the saphenous vein was macroscopically photographed with a digital camera. According to LacZ transgene expression obtained on saphenous vein sections 2 hours after virus exposure, both fiber 16 and fiber 51 chimeric viruses give higher infection. This is because far more blue staining is observed with these viruses compared to Ad5.LacZ (FIG. 8e). The same experiment described for the saphenous vein was performed on human pericardium and human coronary arteries (RCA and LAD). The results of these experiments (Figures 8f, 8g, and 8h, respectively) confirm that the fiber 16 and 51 mutants are superior to Ad5 for infection of human cardiovascular tissue, along with experiments performed on primary cells. It was.
[0082]
E) CAR and integrin expression in human EC and SMC
From the above results, it is clear that chimeric adenovirus with fiber 16 derived shaft and knob is well suited for infecting endothelial cells and smooth muscle cells. Therefore, by changing the fiber protein on the Ad5 virus, we can increase the infection of cells that are difficult to infect Ad5. Although the difference between Ad5 and Ad5Fib16 is significant in both cell types, it is less pronounced in endothelial cells than in smooth muscle cells. This may reflect differences in receptor expression. For example, HUVsmc is significantly more α than HUVEC_vβ5 integrin is expressed (see below). Alternatively, this difference may be due to differences in fiber 16 receptor expression. Ad5.LucFib16 infects umbilical vein smooth muscle cells about 8 times better than umbilical vein endothelial cells, whereas Ad5.Luc virus infects endothelial cells better than smooth muscle cells. To investigate whether Ad5 infection correlates with receptor expression in these cells, CAR and α_vThe presence of integrin was assayed with a flow cytometer. To that end, 1 × 10^FiveAfter single HUVEC cells or HUVsmc were washed once with PBS / 0.5% BSA, the cells were pelleted by centrifugation at 1750 rpm for 5 minutes at room temperature. Next, the cell pellet was diluted 100 times with α._vβ3 antibody (Mab 1961, Brunswick chemie, Amsterdam, The Netherlands) 10 μl, α diluted 100-fold_vβ5 antibody (Mab 1976, Brunswick chemie, Amsterdam, The Netherlands) or CAR antibody diluted 2000-fold (given by courtesy of Dr. Bergelson, Harvard Medical School, Boston, USA) (Hsu) And the like) were added, and the cells were incubated at 4 ° C. for 30 minutes in the dark. Following this incubation, the cells were washed twice with PBS / 0.5% BSA and pelleted again by centrifugation at 1750 rpm for 5 minutes at room temperature. To label the cells, 10 ml of rat anti-mouse IgG1 labeled with phycoerythrin (PE) was added to the cell pellet and immediately the cells were again incubated at 4 ° C. for 30 minutes in the dark. Finally, the cells were washed twice with PBS / 0.5% BSA and analyzed with a flow cytometer. The results of these experiments are shown in Table III. From these results, it can be concluded that HUVsmc does not express detectable levels of CAR, confirming that these cells are difficult to transduce with adenoviruses that enter the cell through the CAR receptor. It was.
[0083]
F) Infection of human A549 cells
As a control for experiments performed on endothelial cells and smooth muscle cells, confirm that equal amounts of viral particles of various chimeric adenoviruses show significant differences in transgene expression in cell lines readily infected by adenovirus In order to do so, A549 cells were infected. This is to examine whether the difference in infection efficiency observed between endothelial cells and smooth muscle cells is cell type specific. 10 for that^FiveA549 cells were seeded in a 24-well plate in a volume of 200 μl. Two hours after sowing, the medium was replaced with medium containing various amounts of fiber chimera 5, 12, 16 or 40-L particles (MOI = 0, 5, 10, 25, 100, 500). 24 hours after virus addition, cells were washed once with PBS, then lysed by adding 100 μl of lysis buffer to each well (1% Triton X-100 in PBS), and then transgene expression The amount (luciferase activity) and protein concentration were determined. Next, the luciferase activity per 1 μg of protein was calculated. The data shown in Table IV demonstrates that Ad5.Luc virus most efficiently infects A549 cells and Ad5LucFib16 or Ad5LucFib40-L infection efficiency is several times lower. This means that efficient infection of endothelial cells and especially smooth muscle cells is due to differences in virus binding to these cells, not the amount or quality of the virus used.
[0084]
[Table 1]

[0085]
[Table 2]

[0086]
[Table 3]

[0087]
[Table 4]

[0088]
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[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a schematic of the pBr / Ad.Bam-rITR construct.
FIG. 2 is a schematic of the method used to delete the fiber gene from the pBr / Ad.Bam-rITR construct.
FIG. 3 is a schematic representation of the construct pBr / Ad.BamRΔFib.
FIG. 4A is a sequence of fiber Ad5.
FIG. 4B is a sequence of chimeric fiber Ad5 / 12.
FIG. 4C is a sequence of chimeric fiber Ad5 / 16.
FIG. 4D is a sequence of chimeric fiber Ad5 / 28.
FIG. 4E is the sequence of chimeric fiber Ad5 / 40-L.
FIG. 5 is a schematic diagram of the construct pC1ipsal-Luc.
FIG. 6 is a schematic diagram of a method for producing a chimeric adenovirus using three overlapping fragments. An initial region (E) and a late region (L) are shown. L5 is a fiber coding sequence.
FIG. 7 a) Infection of HUVEC cells with different amounts of viral particles and different fiber chimeric adenoviruses per cell. Virus concentration: 10000 vp / cell (= white bar), 5000 vp / cell (= gray bar), 2500 vp / cell (= black bar), 1000 vp / cell (light gray bar), 250 and 50 vp / cell are background No detectable luciferase activity above. Luciferase activity is expressed in relative luminescence units (RLU) per microgram of cellular protein.
FIG. 7 b) B) HUVEC cells using different concentrations of cells (22500, 45000, 90000 or 135000 cells seeded per well) and adenovirus serotype 5 (black bars) or fiber 16 chimeric adenovirus (white bars). Infection. Luciferase activity is expressed in relative luminescence units (RLU) per microgram of cellular protein.
FIG. 7c. Flow cytometric analysis of human aortic ECs transduced with 500 viral particles (black bars) or 5000 viral particles (grey bars) Ad5 or Fiber 16 chimeric virus (Fib16) per cell. Non-infected cells were used to set the background to 1%, a median fluorescence value of 5.4. The maximum shift in the central fluorescence value that can be observed with a flow cytometer is 9999. The latter indicates that both Ad5 and Fib16 are 5000 vp / cell and are outside the sensitivity scale of the flow cytometer.
FIG. 8 a) Infection of HUVsmc cells with different amounts of virus particles per cell and different fiber mutant Ad5-based adenoviruses. Virus concentration: 5000 vp / cell (= white bar), 2500 vp / cell (= gray bar), 1250 vp / cell (= dark gray bar), 250 vp / cell (= black bar) or 50 vp / cell (= light gray) rod). Luciferase activity is expressed in relative luminescence units (RLU) per microgram of cellular protein.
FIG. 8b. B) HUVsmc cells using different concentrations of cells (10000, 20000, 40000, 60000 or 80000 cells per well) and adenovirus serotype 5 (white bars) or fiber 16 chimeric adenovirus (black bars). Infection. Due to the fact that the expression of the transgene is higher than the sensitivity range of the bioluminescence meter used, a peak is observed after infection with chimeric fiber 16 adenovirus.
FIG. 8 c) Human umbilical vein SMC transduced with 500 vp / cell (black bars) or 5000 vp / cell (grey bars) using Ad5 or fiber 16 mutant (Fib16). Non-transduced cells were used to set the background median fluorescence value to approximately 1. The central fluorescence value of GFP expression measured by a flow cytometer is shown.
FIG. 8d) D) HUVsmc per cell 312 (light gray bar), 625 (gray bar), 1250 (black bar), 2500 (dark gray bar), 5000 (light gray bar) or 10000 ( White bars) virus particles were infected with fiber 11, 16, 35 or 51 chimeric viruses. Luciferase transgene expression, expressed as relative luminescence units (RLU) per microgram of protein, was measured 48 hours after virus exposure.
FIG. 8e. E) Macroscopic sketch of LacZ stained image in saphenous vein specimen. Nuclear target LacZ (ntLacZ) appears deep blue, black or dark gray on black and white printing.
FIG. 8f) Macroscopic photographs of LacZ stained images in pericard specimens. Nuclear target LacZ (ntLacZ) appears deep blue and black in black and white printing.
FIG. 8g. G) Sketch of a macroscopic photograph of a LacZ stained image in a right coronary artery specimen. Nuclear target LacZ (ntLacZ) appears deep blue, black on black and white printing.
FIG. 8h. H) Sketch of LacZ staining on left artery descending (LAD) specimen. Nuclear target LacZ (ntLacZ) appears deep blue and black in black and white printing.
FIG. 9A shows a sequence containing a gene encoding an adenovirus 16 fiber protein published in Genebank and a gene encoding a fiber of an adenovirus 16 variant isolated in the present invention; The fiber protein sequence is from the NdeI site. FIG. 9A Nucleotide sequence comparison.
FIG. 9B is also a nucleotide sequence comparison.
FIG. 9C. Amino acid comparison.

Claims (11)

A gene delivery vehicle provided with a tissue tropism for at least smooth muscle cells and / or endothelial cells, wherein the adenovirus is selected from the group consisting of adenovirus 11, adenovirus 16, adenovirus 35 and adenovirus 51. A gene delivery vehicle which is a recombinant adenovirus of serogroup derived from subgroup C containing a nucleic acid encoding a capsid containing a viral fiber protein , wherein the capsid protein derived from subgroup C adenovirus is a non-fiber capsid protein A gene delivery vehicle .   The vehicle according to claim 1, wherein the serotype derived from group C is adenovirus 5.   The vehicle according to claim 1 or 2, comprising a nucleic acid encoding a capsid comprising the fiber protein of adenovirus 16.   The vehicle according to any one of claims 1 to 3, wherein the recombinant adenovirus comprises an adenovirus nucleic acid modified to reduce or eliminate the ability of the adenovirus nucleic acid to replicate in target cells.   The vehicle according to any one of claims 1 to 4, further comprising at least one non-adenovirus nucleic acid.   At least one said non-adenovirus nucleic acid is apolipoprotein, nitric oxide synthase, herpes simplex virus thymidine kinase, interleukin-3, interleukin-1α, angiostatin and other (anti) angiogenic proteins, antiproliferative proteins, smooth A gene selected from the group of genes encoding myocyte anti-migratory protein, vascular endothelial growth factor (VGEF), basic fibroblast growth factor, hypoxia-inducing factor 1α (HIF-1α) or PAI-1. The vehicle according to claim 5. A method for gene delivery to smooth muscle cells and / or endothelial cells, comprising the step of supplying the gene delivery vehicle according to any one of claims 1 to 6 to smooth muscle cells and / or endothelial cells in vitro .   A construct pBr / Ad. Which contains the adenovirus 5 sequences 21562-31094 and 32794-35938 and further contains the adenovirus 16 gene encoding the fiber protein. BamRfib16.   The adenovirus 5 sequences 21562-31094 and 32794-35938, the adenovirus 16 gene encoding the fiber protein, and the only non-adenovirus sequence backbone of the construct near the right end repeat sequence of adenovirus 5 PBr / AdBamR.pac / fib16 containing the PacI site of   A construct pWE / Ad. Containing the adenovirus 5 sequences 3534-31094 and 32794-35938 and further comprising the adenovirus 16 gene encoding the fiber protein. AflIIrITRfib16.   A construct pWE / Ad. Containing the adenovirus 5 sequences 3534-22443, 2403-331094 and 32794-35938, and further comprising the adenovirus 16 gene encoding the fiber protein. AflIIrITRDE2Afib16.

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