JPH09500782A - Adenovirus vector containing DNA encoding lung surfactant protein - Google Patents

Abstract

  (57) [Summary] An adenovirus belt containing a DNA sequence encoding one lung surfactant protein. This adenovirus vector is a replication-defective adenovirus vector lacking at least many E1 and E3 DNA sequences. Such vectors can be used to generate infectious viral particles that "in vivo" transduce lung epithelial cells to allow expression of lung surfactant proteins such as by lung epithelial cells.

Description

Detailed Description of the Invention           DNA encoding lung surfactant protein           Adenovirus vector containing   This application was filed on April 8, 1994, with application number 08 / 044,406. It is a partial inheritance.   This invention relates to adenovirus vectors. More specifically, this invention An adenovirus vector containing a DNA encoding a surface-active protein, Pulmonary surface-active proteins such as infant respiratory distress syndrome and adult respiratory distress syndrome are also included. Use of such vectors to treat disease states associated with protein loss About things.   Detergent proteins are naturally produced in alveolar cells and airway epithelial cells of normal lung. Phospholipid is a natural endogenous protein that maintains patency of the alveolar structure. Interact with. Concentration of pulmonary surfactant protein on the alveoli surface drops to critical levels Then, the interfacial tension of the liquid-gas interface (interface) increases, which causes It leads to alveolar collapse, inadequate lung aeration-perfusion combination, and hypoxia. Terrible If not, death may occur. Intermittent administration of exogenous bovine lung surfactant protein Shows a partial, but not complete, pathophysiological relief of the surfactant deficiency state ing.   It is possible to provide a recombinant expression vehicle for expressing lung surfactant protein. Is one of the objects of the invention.   Furthermore, it is related to the condition of clinical surfactant protein deficiency and the accompanying gas exchange. In order to suppress the pathophysiological effects of It is also an object of the present invention to provide an expression carrier that can be pulled. is there.   The above objects and others will be made clear in the following specification.   According to one aspect of the present invention, a DNA encoding a pulmonary surfactant protein One adenovirus vector containing the sequences is provided.   In one embodiment, the adenovirus vector is a replication defective adenovirus vector. It is. That is, the vector is, for example, an E1 DNA sequence or a part thereof. Lacks the DNA sequences required for viral replication, such as. In one embodiment And the adenovirus vector contains at least the adenovirus E1 DNA sequence. And lacks at least a portion of the adenovirus E3 DNA sequence. Have been. E3 region helps adenovirus evade host immune surveillance Encodes several polypeptides.   In one embodiment, the adenovirus vector is adenovirus 5'reverse terminal. Terminal repeats, ie ITR, adenovirus 3'ITR, adenovirus envelope sig Null, a DNA sequence encoding a lung surfactant protein, and a lung surfactant protein. It consists of a promoter that controls the expression of the DNA sequence encoding the protein. this The vector lacks at least a large number of E1 and E3 DNA sequences, but E2 And not all of the E4 DNA sequence, and also the adenovirus major late phase Professional DNA sequence encoding an adenovirus protein expressed by a motor Is not lacking. In one embodiment, this vector also contains E2 and E4.   At least one DNA sequence selected from the group consisting of DNA sequences At least some are missing. In another embodiment, the vector is adenovirus. Lacking the Ils E1 and E3 DNA sequences, and E2 and E4 DNA Lacking one of the sequences, and of DNA sequences other than the E2 and E4 DNA sequences. Lacks some.   In a further embodiment, the vector comprises at least a large number of E1 and E3. Lacking DNA sequences, selected from the group consisting of E2 and E4 DNA sequences Is lacking at least part of at least one DNA sequence Adenovirus protein expressed under the control of viral late promoter It lacks the quality-encoding DNA sequence.   DNA sequences encoding lung surfactant proteins are controlled by appropriate promoters Below. Suitable promoters used include, but are not limited to, Adenovirus promoters such as the denovirus major late promoter; Or a heterologous promoter such as the salivary gland (CMV) promoter; Sarcoma virus promoter; Respiratory syncytial virus promoter; Mouse milk Cancer virus or an inducible promoter such as the MMTV promoter; The tarothionein promoter; and the heat shock promoter. In addition Textile-specific promoters, that is, not necessarily so Although not shown, a pulmonary surfactant protein promoter or the like can also be used. I However, the scope of the present invention is not limited to any specific promoter. Must be understood.   Pulmonary interface encoded by a DNA sequence encoding a pulmonary surfactant protein The active proteins are surfactant protein A (SPA), surfactant protein B ( SPB), and surfactant protein C (SPC).   Surfactant protein A is described in Kuroki et al., “Biochemical Journal”, Volume 263, No. 7, 33. 88-8394 (March 5, 1988). Surfactant tongue Protein B and the DNA encoding it are Pilot-Mathias et al. “DNA”. Volume 8, Issue 2, pages 75-86 (1989), Grasser et al. "Bulletin of Me" 84, 4007-4011 (June 1987), Reebok et al. "Clinical Research Journal" Vol. 81, pp. 826-833 (March 1988), Oh Riley et al., "Bio-Kimika-Eto-Biophysica-Acta", 1011, 14 Pp. 0-148 (1989); and Weaver et al. Journal, pp. 982-987 (1988). Surfactant Protein C is further described by Grasser et al., "Biochemistry Journal", Volume 263, No. 21, 10 326-10331 (July 25, 1988).   In one embodiment, the DNA sequence encoding the pulmonary surfactant protein is the lung sequence. Encodes surface-active protein B. Current Evidence suggests that among the above, SPB is the most clinically important lung It is a surfactant protein.   In a preferred embodiment, such a vector is shuttled according to standard procedures. It is first assembled by constructing a plasmid. This shuttle plasmid Including the left adenovirus genomic element, the adenovirus 5'ITR, The "critical left end containing the adenovirus envelope signal and the E1a enhancer sequence. End element ": (adenovirus promoter or foreign promoter) Promoter; tripartite leader sequence, multiple cloning site; poly A signal; And a DNA segment corresponding to a segment of the adenovirus genome You. This DNA segment is homologous using a modified or mutant adenovirus Serves as a substrate for selective recombination, and such a sequence is for example adenovirus. One of the adenovirus genomes from base 3328 to base 6241 of Rus 5 genome It can contain one segment. This plasmid also contains selectable markers and And the origin of replication. The origin of replication can be, for example, a bacterial origin of replication. This A representative example of such a shuttle plasmid is pAVS6 shown in FIG. Introns are added within the transcript to increase the level of cytoplasmic mRNA accumulation Sometimes.   Multiple cloning sites are located in the DNA sequence encoding the lung surfactant protein. Promotes insertion into plasmids. DNA coding for lung surfactant protein Rows can be inserted at multiple cloning sites. Shuttle plasmids in general Of the above The restriction enzyme sites that separate the fractions include "rare" restriction enzyme sites. This "rare" limitation About 1 enzyme site per 10,000 base pairs to about 1 per 100,000 base pairs It is a site found to randomly appear in eukaryotic genes with a frequency of up to. this is The flexibility and rearrangement of the components of the vector in the assembled shuttle plasmid Increase ease.   Homologous recombination is then carried out with the modified or mutant adenovirus, Here, at least a large number of E1 and E3 adenovirus DNA sequences are shown in FIG. It is deleted as shown in. Such homologous recombination results in CaPO_FourTo precipitation Shuttle plasmids to helper cell lines such as 293 (embryonic kidney epithelial) cells It is carried out through co-transfection of adenovirus and modified adenovirus. this Upon homologous recombination, one cloning vector is formed in which the modification vector D on a shuttle plasmid corresponding to a similar segment of the adenovirus genome A modified adenovirus DNA that is 5'to the NA segment has It is replaced with a component on the shuttle plasmid that is 5'to the A segment. This Homologous recombination, or "crossover," events in the modified adenovirus genome segment Can occur anywhere along the Adenovirus 5 base pairs 3328 to 6241 found in Example 1 Corresponding to the segment also included in the shuttle plasmid.   Through such homologous recombination, one vector is formed. Adenovirus 5'ITR; Adenowi Rus envelope signal; E1a enhancer sequence; promoter; tripartite leader sequence Lane; DNA sequence encoding lung surfactant protein; poly A signal; less Adenovirus lacking both E1 and E3 adenovirus DNA sequences DNA; and adenovirus 3'ITR. This vector is then In order to produce high amounts of infectious recombinant adenovirus particles, such as the 293 cell line Introduced into the cell line. The 293 cell line is a human fetal kidney epithelial cell line against which The left end 11% of the adenovirus 5 genome was introduced unchanged. This is adeno Directed the synthesis of viral E1a and E1b proteins and transferred the E1 deletion vector. Allows transcomplementation.   Infectious viral particles are then hosted "in the maternity period" as part of gene therapy treatment Is administered. The infectious viral particles may be injected intravenously or intramuscularly or subcutaneously. It is administered systemically, such as by administration, or locally, such as by intratracheal or intrabronchial administration. May be administered locally or, alternatively, infectious viral particles may be an aerosol formulation. It can be administered in the form. Infectious virus particles are about 10¹³up to pfu , Preferably about 10⁷about 10 from pfu¹²It is administered in amounts up to pfu. For example Infectious viral particles are used to transduce airway or alveolar epithelium. this This allows lung epithelial cells to achieve sufficient clinical correction of lung surfactant protein deficiency. Express lung surfactant protein in modest amounts.   In addition, infectious viral particles are used to transduce eukaryotic cells "in vitro". Can be used. Transduced eukaryotic cells Vesicles include, but are not limited to, macrophages, lymphocytes, fibroblasts, Includes hepatocytes, bronchial cells, and other epithelial or endothelial cells. True like this The nuclear cells are then administered to the host as part of a gene therapy treatment, or Cells can be cultured "in vitro" to produce lung surfactant protein I can do it.   Moreover, infectious viral particles are responsible for the "in vitro" production of lung surfactant proteins. Therefore, it can be used to transduce eukaryotic cells "in vitro". Lung field True transduced "in vitro" for "in vitro" production of surface-active proteins Examples of nuclear cells include, but are not necessarily limited to, eukaryotic cells described above, and Chinese hamster ovary (CHO) cells, COS-7 cells, NIH 3T3 cells, vero cells, HeLa cells, MRC-5 cells, CNl cells, W138 cells and Contains chicken lymphoma cells. Pulmonary surfactant protein produced by such cells Then with an acceptable pharmacological carrier to treat the pulmonary surfactant protein deficiency condition It is administered to the host in combination.   In another embodiment, the vector is adenovirus 5'ITR; adenovirus. Rus 3'ITR; adenovirus envelope signal; encodes pulmonary surfactant protein Sequence that encodes a pulmonary surfactant protein, and a DNA sequence that encodes a lung surfactant protein. It consists of a promoter. The vectors are adenoviruses E1, E2, E3 and And the E4 DNA sequence, and the vector contains the adenovirus major late DNA sequence encoding adenovirus protein promoted by a motor Missing Have been. That is, the vector D encodes an adenovirus structural protein. It lacks NA. Such vectors are sometimes referred to below as "Gutless adenovirus. "Vector" (an adenovirus vector without content) or "GLAd" vector. Quoted as the actor.   The promoter contained in the vector is as described above.   Such a vector can be derived from the adenovirus genome using standard techniques. 5'ITR, adenovirus 3'ITR, and adenovirus envelope signals Can be removed and constructed. Such ingredients, likewise (adenovirus pro Motor or non-adenovirus promoter) , The tripartite leader sequence, the poly A signal can be isolated by standard techniques, eg pKSII (Stratagen, Inc.) and other base plasmids or “starter” plasmids. It can be ligated into a rasmid to form a suitable cloning vector. Ku The rolling vector contains the multiple cloning site described above and contains the foreign DNA Facilitates insertion of the column into the cloning vector. One suitable according to this invention Of the vector is thus compatible with standard techniques with appropriate restriction sites for multiple cloning sites. Cleaves the cloning vector and then encodes a pulmonary surfactant protein Is formed by ligating the DNA sequence of   The GLAd vector is then a helper add-on that provides the necessary packaging materials. Infectivity using noviruses or cell lines Packaged in viral particles. Helper virus was used in one example In some cases, the helper virus will prevent the helper virus from packaging itself. It is desirable that the louse have a defective envelope signal. So that can be used Examples of various envelope-defective helper viruses include Greyble et al., “Virology Journal” 6 6, pp. 723-731 (1992), and Grable et al., "Virology. Journal ”, 64, 2047-2056 (1990). You. In another embodiment, the helper virus is a normal packaging signal. It is.   The DNA for the vector and the envelope defective helper virus is an infectious virus. The appropriate cell line is transfected for the production of particles. Transfection is electroporation, Calcium citrate precipitation, microinjection or through proteoliposomes Koru Examples of suitable cell lines include, but are not necessarily limited to, HeLa cells, A54 9 cells, or 293 (embryonic kidney epithelium) cells. Infectious virus particles are next Purified from the helper virus by CsCl isopycnic centrifugation, as described above. Transduced into lung epithelial cells lining the respiratory tract or alveoli, which The cells express lung surfactant protein.   In another alternative, the vector is transfected into cells and the enveloped defective helper virus is Followed by infection of the cells with cells.   The invention will be described in connection with the following examples, the scope of which is It is not limited.   Example 1   Adenovirus construction shuttle plasmid pAvS6 based on cloning technique Several steps using standard cloning techniques involving the polymerase chain reaction. Built with First, the 2913 base pair BglII and HindIII fragments were Ad-d. PKSII (La Jolla, Calif.) As a blunt fragment. , Stratagen) at the XhoI site (FIG. 1). Ad-d132 7 (Zinmapappya et al., "Cells", Vol. 31, 543 (1983)) Denovirus 5 genome base pairs 28593 to 30470 (or figure unit 78 . 5 to 84.7) and the deletion of the XbaI fragment, which is located in the E3 region Adenovirus 5 except that   Complete adenovirus 5 genome registered at GenBank Access # M73260 , Which is incorporated herein by reference, and the virus Maryland, Rockville, American Type Culture Collection Can be used with access number VR-5.   Ad-d1327 was constructed from adenovirus 5 (Ad5) by conventional methods. Was. This method is briefly outlined below and was previously described by Jones and Schenck. "Cells" Vol. 13, pp. 181-188 (1978). Ad5 DNA was isolated by proteolytic digestion of virions and It is partially cleaved by the endonuclease. The Xba I fragment is then a fragment Are reconstituted by conjugation as a mixture of As a result, from the sequence 28593 base pairs Has a sequence similar to Ad5 except that it removes up to 30470 base pairs Yields several attached genomes. This DNA is then used in suitable cells (eg KB cells, HeLa cells and 293 cells can be transfected to form plaques Sea urchin agar is added. Individual plaques were then separated and amplified, 1878 Screened in the absence of the base pair E3 region Xba I fragment. A fragment of this fragment The BglII site is oriented relative to the T7 RNA polymerase site of pKSII. And the HindIII site is near the T3 RNA polymerase part of pKSII. It was shaped so that it was closest to the rank. This plasmid is named pHR (Fig. 1).   Second, ITR, envelope signal, Rous sarcoma virus promoter, adenovirus The Ils tripartite leader (TPL) sequence and the linked sequence were cloned using PCR amplification. (Figure 2). ITR and envelope signals ([GenBank, Access # M73260 sequence identical to Ad5 sequence] Ad-d1327 sequence 1-392) uses primers containing NotI or AscI restriction sites to Co-amplified from d-d1327 (amplification 1). Rous sarcoma virus LTR The lomotor is positive with primers containing AscI and SfiI sites. Mido pRC / RSV (Sequences 209-605; California, San Diego, Amplified from Invitrogen (amplification 2). DNA from amplifications 1 and 2 The product uses only NotI and SfiI primers Ligated using the "wrap" PCR method (amplification 3). Reaction 1 and Amplification of complementation between AscI-containing ends of each initial DNA amplification product obtained from In time allowed the conjugation of these two parts. The TPL is then SfiI and 16 hours with Ad-d1327 using primers containing each XbaI site Amplified from cDNA isolated from mRNA isolated from 293 infected cells. Widened (amplification 4) ([GenBank, Access # M73260 from Ad5 Identical to similar sequences] Ad-d1327 sequences 6049-9730). Amplification reaction The DNA fragments obtained from 3 and 4 then had NotI- and XbaI-sites. Was ligated using PCR with the included primers (amplification 5) and thus the complete gene block Created.   Third, the ITR envelope signal-TPL fragment was subsequently purified, NotI and Cleaved with XbaI and inserted into NotI and XbaI digested pHR plasmid Was. This plasmid was named pAvS6A and its orientation is NotI of this fragment. The site was made to flank the T7 RNA polymerase site (Figure 3).   Fourth, the SV40 early poly A signal is as an HpaI-BamHI fragment. PAvS6 removed from SV40 DNA and treated with T4 DNA polymerase SalI of plasmid pAvS6A- (FIG. 3) to create (FIGS. 3 and 4) Was inserted into the site.   2 kilobase DNA containing whole human lung surfactant protein B (SPB) cDNA The fragment (Fig. 5) (Pilot-Mathias, 1989) was digested with EcoRI and Plasmid Obtained from pKC4-SPB (Fig. 6) (Weaver et al. Journal ”, pages L-95 to L-103 (1992)). This DNA fragment is Separated, purified, and then cloned into the EcoRV site of plasmid pAVS6. (Fig. 4). Three identical clones with correctly inserted SPB cDNA were obtained. Was. The clones are pAVS6 / SPB # 7, pAVS6 / SPB # 12, And pAVS6SPB # 13. Figure 7 shows pAVS6 and SPB # 7. Indicated by. The orientation of SPB DNA in the shuttle plasmid is from pAVS6 Introduce the two ends of the SPB cDNA insert into the plasmid with the derived primers. Obtained by evaluating the DNA sequence.   Recombinant adenovirus vector AV1SPB1 containing SPB cDNA (FIG. 8). ) Is the Ad5 deletion mutant Ad-d1327 (FIG. 8) and pAVS6.SP. It was constructed through homologous recombination between B # 7. Homologous recombination, or "transfer "E" means bases 3328 to 6241 (or figure units) of the adenovirus 5 genome. Ad-d1327 and pAVS6.SP corresponding to 9.24 to 17.34) Ad-d1327 and pAVS6 SPB along with the segment common to B # 7 It occurs with # 7. Ad-d1327 does not have base pairs 28593 to 30470. Has a deleted E3 region (Zinmapappa et al., "Cells" 31: 543 -551 (1982)). pAVS6 / SPB # 7 is an adenovirus An origin of replication completely contained within the 5'ITR, 5'ITR, E1a enhancer and envelope signal, Rous sarcoma virus promoter, Denovirus 5 tripartite leader sequence, and the entire protein coding sequence (nucleus 2 kilobase human SPB cDNA containing Otide 1 to 1172) and S Contains the V40 poly A signal.   Example 2   293 cells (ATCC No. CRL1573) had a multiplicity of infection of 50 MOI units ( MOI) infected with AV1SPB1. 12 hours after infection, the cells³⁵S Methio Radiolabeled with nin (50 μCi / ml) overnight. The same amount of labeled protein Antiserum to SPB was used for immunoprecipitation. Immunoprecipitates are SDS poly It was analyzed on a 16% gel by acrylamide gel electrophoresis. Fluorescence of gel Was done. C¹⁴Labeled molecular weight markers and bio-rad broad molecular weight markers It was used as a zoomer. As shown in Figure 9, lane 1 represents the uninfected control. Shown in lane 2 is AV1SPB1 where electrophoresis of immunoprecipitates occurs under reduced conditions. Infected 293 cells are shown, and lane 3 is a non-reduced state of immunoprecipitation electrophoresis. Shown are AV1SPB1 infected 293 cells. Active SPB peptide is about Mr = 6,000 to 8,000 (reduced state), and naturally occurring human SPB (Mr = It forms an oligomer (non-reduced state) identical to that of 18.000). Precursor Protein quality was detected in both reduced and non-reduced conditions.   ExampleThree   Mouse lung type II epithelial cell line is an adenovirus vector The AV1SPB1 was transduced with the Rous sarcoma virus. Includes full length human SPB cDNA under control of the promoter. SPB expression is within Specific for causative mouse SPB (upper band) or human SBP (middle band)³²P Assayed by ribonuclease protection using labeled probe. B tube Phosphorus-specific probes (bottom band) also ensured that the same amount of RNA was added to each assay. Used to be. The B tubulin probe is the human B tuin in lane 1. -I didn't recognize Bryn. As shown in FIG. 10, cells have 50 multiplicities of infection ( MOI) units (lane 2), 100 MOI units (lane 3), also 150 MOI Unit (lane 4) was infected with Av1SPB1 and apparently emits human SPB mRNA. Revealed. Human SPB was not detected in uninfected cells (lane 5).   Example 4   Four cotton rats were anesthetized with metaphen, 1.5 x 10^Tenpfu Given Av1SPB1. For rats, 2 days (n = 1 animal) and 3 days (n = 2) after administration , Or 45 days (n = 1) and the lungs were harvested. Whole lung RN A is the guanidine thiocyanate-CsCl method (Charguin et al., “Biochemistry”) 18: 5294-5299 (1979)). all Lung RNA (15 μg) was subjected to formaldehyde agar gel electrophoresis and Transferred to membranes (Nitran, Schleicher and Schul). Filter crosslinked (Stratagen, UV cross-linked), random prime (loft strand Company) To³²Hybridized with P-labeled 2.0 kilobase human SP-B cDNA probe Formed and evaluated by autoradiography. Uninfected control rats and And lungs from rats infected with Av1LacZ4 were also treated with said hybridization. Received.   Northern blotting analysis shown in Figure 11 shows that human SP-B mRNA Expressed in the lungs of cotton rats infected with Av1SP-B1 but not in the lungs of uninfected animals. That it was not expressed in the lungs of animals infected with Av1LacZ4. Was showing. 2.0 kilobase mRNA for human SP-B gene is Av1 Detected in cotton rats infected with SP-B1, which was transcribed by the vector And the size of full-length human SP-B mRNA.   Example 5   Two cotton rats were anesthetized with metaphene (10 with PBS).^Tenpfu / Av1SP-B1 (diluted to 300 μl) was administered by intranasal instillation. Feeling Two days after staining, the rats were sacrificed and the lungs were harvested. The lungs were washed twice with PBS and no Perfused with methionine LHC-8 medium (Biofluid), finely divided,³⁶ Incubate in S-Cys / Met (medium 1 ml, 100 μCi / ml) medium for 24 hours. I was warmed. The presence of SP-B in medium and lysed lung explants resulted in secreted SP-pepti Rabbit anti-human SP-B antiserum for detecting SP and B-treated peptides Was evaluated by an immunoprecipitation method using (Weaver et al. “American Physiology Journal”). Le 263, L95- L103 page (1992)). Av1LacZ4 treated rats and untreated rats Was used as a control.   As shown in FIG. 12, the “denovo” synthesis of human SP-B peptide (new (Regular synthesis) and secretion from lung fragments removed from animals infected with Av1SPB1 Detected and detected in non-infected animals or animals infected with Av1LacZ4 Was not done. Human SP-B peptide is 8 and 18 kilodaltons Secretion was detected in an oligomeric form of the precursor SP-B (pro-SP-B). ) Derived vector is "in vivo" adenovirus vector-mediated gene transfer It was suggested that it was processed after.   Example 6   The cotton rat has Av1SP-B1 (a) 0.5 × 10 in the nasal cavity.⁹pfu (n = 1); (b) 1.5 × 10⁹pfu (n = 1): or (c) 1.5 × 10^{1 0} Treated with an amount of pfu (n = 1). The animals were sacrificed 48 hours after infection , Wort et al., "Evolutionary Biology", Vol. 156, 426-443 (1993) In accordance with the method, the lungs were hybridized "in situ" with human SP-BcRNA. Prepared by the pad forming method. Uninfected rats were used as controls.   The lungs were swollen overnight at 4 ° C in 4% paraformaldehyde in 20 cm of water. fixed. Radiolabeled Libroprobe for human SP-B³⁵S-VTP Produced by "in vitro" transcription in the presence. Slides are human S under stringent conditions Hybridized to P-B probe Exposed to mulsion, then slides are post-stained with hematoxylin and eosin Was done. Antisense and sense probes are Av1SPB1 treated, Av1La Comparisons were made in cZ4 treated and control (untreated) animals.   "In situ" hybridization of Av1SP-B1 infected cotton rats Expression of human SP-B mRNA in bronchiolar and alveolar epithelial cells was shown. . Human SP-B mRNA was detected in patchy distribution and is shown in FIGS. 13 to 16. As shown in the figure, these figures show that Av1SP-B1,0. 5 × 10⁹pfu; Av1SP-B1, 1.5 × 10⁹pfu; Av1SP-B1 , 1.5 × 10^TenRats treated with pfu; and uninfected control rats, respectively "In situ" hybridization results of Av1SP-B1 Light microscopic analysis of lungs of animals treated with Av1LacZ showed that peribronchiolar lymphocytes Mild inflammatory response in mononuclear cell infiltrate, increased microphages, and some Polymorphonuclear leukocytes were shown. The infiltrate was remarkable after 48 hours and was dose-dependent . In parallel experiments with Av11acZ4, the infiltrate was 3-4 weeks after exposure. Basically disassembled.   All patents, published information and databases cited in this specification are The state of the art of the person skilled in the art of the invention is indicated. Disclosure of such patents, published Published information, including patent applications, and database entries are Each of the patents, published information and database entries of Indicate specifically and individually that it should be incorporated To the same extent as if quoted as a whole here It is included.   However, it is understood that the scope of the present invention should not be limited to the above embodiment. Must be done. The present invention can be carried out in addition to those specifically described. It is within the scope of claims.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI C12P 21/02 9051-4C A61K 37/00 // (C12P 21/02 C12R 1:91) (72) Inventor Trapnell, Bruce United States, 20895 Maryland, Kensington, Bird Road 4023 (72) Inventor Whitset, Jeffrey United States, 45230 Ohio, Cincinnati, Salem Road 5565

Claims (1)

[Claims] 1. An adenovirus vector containing a DNA sequence encoding one lung surfactant protein. 2. The vector according to claim 1, wherein the lung surfactant protein is surfactant protein B. 3. The vector according to claim 1, wherein the adenovirus vector is a replication-defective adenovirus vector. 4. 4. A vector according to claim 3, wherein said adenovirus vector lacks at least a portion of the adenovirus E1 DNA sequence and also lacks at least a portion of the adenovirus E3 DNA sequence. 5. 5. The vector according to claim 4, wherein the vector is: adenovirus 5'ITR; adenovirus 3'ITR; adenovirus envelope signal; DNA sequence encoding a pulmonary surfactant protein; A promoter controlling a DNA sequence, wherein said vector lacks at least a number of adenovirus E1 and E3 DNA sequences, but contains all E2 and E4 DNA sequences, as well as adenovirus proteins controlled by the adenovirus major late promoter. A vector comprising a promoter which does not lack a coding DNA sequence. 6. 6. A vector according to claim 5, wherein said vector lacks at least a number of adenovirus E1 and E3 DNA sequences and at least one DNA selected from the group consisting of E2 and E4 DNA sequences. A vector, characterized in that it lacks at least part of the sequence. 7. 7. The vector of claim 6, wherein said vector lacks at least a number of adenovirus E1 and E3 DNA sequences, and lacks one of E2 and E4 DNA sequences, and further comprises E2 and E4. A vector lacking a part of a sequence other than a DNA sequence. 8. A vector according to claim 7, wherein said vector lacks at least a number of E1 and E3 DNA sequences and comprises at least one DNA sequence selected from the group consisting of E2 and E4 DNA sequences. A vector lacking at least a portion and lacking a DNA sequence encoding an adenovirus protein driven by the adenovirus major late promoter. 9. The vector according to claim 5, wherein the promoter is an adenovirus promoter. 10. The vector according to claim 5, wherein the promoter is a heterologous promoter. 11. An infectious virus particle produced from the vector of claim 1. 12. Lung epithelial cells transfected with the infectious viral particle of claim 11. 13. An infectious virus particle produced from the vector according to claim 5. 14． Lung epithelial cells transfected with the infectious viral particle according to claim 13. 15. A method of treating a pulmonary surfactant protein deficiency condition, comprising administering to a host an effective amount of infectious viral particles according to claim 11. 16. A method of treating a pulmonary surfactant protein deficiency condition, comprising administering to a host an effective amount of infectious viral particles according to claim 13. 17． 16. The method of claim 15, wherein the infectious viral particle is administered in an amount up to about 10 ¹³ pfu. 18. 18. The method of claim 17, wherein the infectious viral particle is administered in an amount of about 10 ⁷ pfu to about 10 ¹² pfu. 19. 17. The method of claim 16, wherein the infectious viral particle is administered in an amount of about 1 pfu to about 10 ¹² pfu. 20. 20. The method of claim 19, wherein the infectious viral particle is administered in an amount of about 10 ⁷ pfu to about 10 ¹² pfu. 21. 16. The method according to claim 15, wherein the pulmonary surfactant protein deficiency condition is infantile respiratory distress syndrome. 22. 17. The method according to claim 16, wherein the pulmonary surfactant protein deficiency condition is infantile respiratory distress syndrome. 23. 16. The method according to claim 15, wherein the pulmonary surfactant protein deficiency state is adult respiratory distress syndrome. 24. The method according to claim 16, wherein the pulmonary surfactant protein deficiency state is adult respiratory distress syndrome. 25. An adenovirus vector, lacking at least a portion of the adenovirus E1 DNA sequence, lacking at least a portion of the adenovirus E3 DNA sequence, and also selected from the group consisting of E2 and E4 DNA sequences An adenovirus vector, characterized in that it lacks at least a portion of at least one DNA sequence described. 26. The vector is characterized in that it lacks the adenovirus E1 and E3 DNA sequences, also lacks one of the E2 and E4 DNA sequences, and further lacks part of the sequences other than the E2 and E4 DNA sequences. The vector according to claim 25. 27. 26. The vector of claim 25, wherein the vector lacks at least a portion of the E2 DNA sequence. 28. 26. Vector according to claim 25, characterized in that said vector lacks at least part of the E4 DNA sequence.