CA2410981A1 - Adenovirus particles with mutagenized fiber proteins - Google Patents

Abstract

This invention relates to mutated adenoviral fiber proteins and adenovirus particles containing such proteins. It further relates to polynucleotides encoding the proteins and vectors containing polynucleotides. It also relates to methods for making and using the adenoviral particles. With the mutated fiber proteins, the adenovirus particles no longer bind to their natural cellular receptor. They can then be "retargeted" to a specific cell type through the addition of a ligand to the virus capsid, which causes the virus to bind to and infect such cell. Specific fiber mutations are listed, which ablate binding to the natural receptor. Adenovirus particles with certain fiber mutations were found to enhance gene transfer to and expression in liver as compared to viral particles with wild-type fiber.

Description

ADENOVIRUS PARTICLES WITH
MUTAGENIZED FIBER PROTEINS
This application claims the benefit under 35 USC ~ 119(e) of the following United States. provisional applications: (1) Provisional Application No. to be assigned, filed June 2, 2000 as Application No. 091585,344, and subject to a Petition for Conversion to Provisional Application filed December 21, 2000; and (2) Provisional Application No.
60/270,555, filed February 22, 2001. The disclosures of these applications are incorporated herein by reference in their entirety.
BACKGROUND OF THE INVENTION
The wide tropism of adenoviral vectors is one of its advantages as a gene=delivery vehicle. However, there are a number of reasons why targeted vectors are desirable.
Adenoviral vectors with increased transduction specificity should show reduced , toxicity, since lower doses could be delivered to achieve the same desired therapeutic benefit. In addition, these lower doses sh= >uld reduce potential immune responses to the viruses. Thi:q ~ .
increased safety of targeted vectors would then allow for new routes of delivery, such as systemic administration, that would be applicable to a number of indications, like cancer and cardiovascular disease. Adenoviral particles with mutagenized fiber proteins are useful in the preparation of targeted adenoviruses.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows the strategy used for the production of pseudotyped adenoviral vectors with transiently expressed fiber proteins using the transient transfection/infection system.
Figuxe 1A shows a schematic diagram of the genomic structure of Ad5.j3gal.OF.
Figure 1B
shows the transient transfection/infection system. The fiber deleted adenoviral vector, AdS.(3gal.dF, as shown in panel A, can be grown in packaging cell lines transiently or stably expressing different fiber proteins to generate Ad5.~3gal.OF/F+ fiber containing adenoviral particles. The vector is used to infect 293T cells that have been transfected with a fiber expression plasmid. The resulting particles will have new receptor tropisms dependent on the fiber protein.
Figure 2 shows the differential fiber-dependent adenoviral transduction properties of HeLa cells using pseudotyped adenoviral vectors. HeLa cells were transduced with 1000 total SUBSTITUTE SHEET (RULE 26) particles per cell with the indicated pseudotyped adenoviral vector. After 24 hours, the cells were analyzed for ~3-galactosidase activity using a chemiluminescence reporter assay. The relative (3-galactosidase activity of each pseudotyped adenoviral vector containing a mutated fiber protein was determined and normalized as a percentage of AdS.(3gal.~F/wt, which contains a wildtype fiber protein. All values are the mean percentage of AdS.
j3gal.OF/wt, ~
standard deviation (sd) derived from 5 to 6 separate transductions.
Figure 3 is a plasmid map for pSFIoxPRGD.
Figure 4 is a plasmid map for pAv 1 hlpr.
Figure 5 is a plasmid map for pSK02, containing fiber mutations in .combination with a cRGD targeting moiety.
Figure 6 shows.-the transduction efficiency of adenovirus. with retargeting.ligand and detargeting fiber mutations. HDF (Fig. 6A), HeLa (Fig. 6B), CHO-Kl (Fig. 6C), and PC3 (Fig. 6D) cells were infected at 20 to 12500 total particles per cell in five-fold dose increments with the indicated fiber-modified adenoviral vectors. AvlnBg is the parental control with an unmodified fiber gene, AvlnBgHTRGD has been genetically altered to include cRGD in the HI loop, AvlnBgHIRGDK01 has been genetically altered to include cRGD in the HI loop and the S408E, P409A mutation in fiber knob, and AvlnBgHIRGDK02 has been genetically altered to include cRGD in the HI loop and the ~V44I, K442 mutation in fiber knob. After 24 hours, the cells were analyzed for 13-galactosidase reporter gene activity using a chemiluminescence reporter assay.
Figure 7 shows that AvlnBgHIRGDK02 can compete transduction of HDF cells with AvIGFPHIRGD. HDF cells were infected at 1000 particles per cell with AvIGFPHIRGD, an adenoviral vector expressing GFP and which has been genetically altered to include cRGD
in the HI loop. The infections were competed with AvlnBg, AvlnBgHIRGD, AvlnBgHIRGDKOI, and AvlnBgHIRGDK02 at doses ranging from 1000 to 128,000 particles per cell in four-fold dose increments. After 24 hours, the cells were analyzed for GFP expression by measuring the fraction of cells that were positive for GFP
expression by FACS analysis. The data was normalized as a percentage of AvIGFPHIRGD without .
competitor.
Figure 8 is a plasmid map for pSK0l, containing fiber mutations in combination with a cRGD targeting moiety.
Figure 9 is a plasmid map of pFLAv3nBgK01 containing the full-length adenoviral genome with the KOI fiber AB loop mutation.

SUBSTITUTE SHEET (RULE 26) Figure 10 shows the transduction efficiency of Hela (Fig. 10A) and HDF (Fig.
10B) cells using adenoviral vectors containing fiber AB loop mutations.
Figure 11 shows the transduction efficiency of Hep3B (Fig. 11A), HepG2 (Fig.
11B) and mouse hepatocytes (Fig. 11C) using adenoviral vectors containing fiber AB
loop mutations.
Figure 12 shows a competition viral transduction assay.
Figure 13 shows irc vivo adenoviral-mediated expression of ~i-galactosidase by an analysis of ~i-galactosidase activity in mouse livers.
Figure 14 shows in vivo adenoviral-mediated transduction of mouse livers by hexon PCR analysis.
_. -. Figure . 15 shows in _. vivo___. adenQViral-mediated expression expression.__,_o.~. .(3-.._. .
galactosidase by an analysis of ~3-galactosidase activity in C57BL/6, Balb/C, and CD-1 mouse livers.
Figure 16 shows ih vitro adenoviral-mediated transduction of isolated primary mouse hepn:~:ocytes.
DESCRIPTION OF THE INVENTION
This invention relates to mutated adenoviral fiber proteins and adenovirus particles containing such proteins. It further relates to polynucleotides encoding the proteins and vectors containing the polynucleotides. It also relates to methods for making and using the adenoviral particles. ~ With the mutated 'fiber proteins, the adenovirus particles no longer bind to their natural cellular receptor. They can then be "retargeted" to a specific cell type through the addition of a ligand to the virus capsid, which causes the virus to bind to and infect such cell.
As used herein, the term "adenovirus" or "adenoviral particle" is used to include any and all viruses that may be categorized as an adenovirus, including any adenovirus that infects a human or an animal, including all groups, subgroups, and serotypes.
Preferably, such adenoviruses are ones that infect human cells. Such adenoviruses may be wild-type or may be modified in various ways known in the art or as disclosed herein. Such modifications include modifications to the adenovirus'genome that is packaged in the particle in order to make an infectious virus. Such modifications include deletions known in the art, such as deletions in one or more of the E1, E2a, E2b, E3, or E4 coding regions. Such modifications also include deletions of all of the coding regions of the adenoviral genome.
Such SUBSTITUTE SHEET (RULE 26) adenoviruses are known as "gutless" adenoviruses. The terms also include replication-conditional adenoviruses; that is, viruses that replicate in certain types of cells or tissues but not in other types. These include the viruses disclosed in U.S. Patent No.
5,998,205, issued December 7, 1999 to Hallenbeck et al. and U.S. Patent No. 5,801,029, issued September l, 1998 to McCormick, the disclosures of both of which are incorporated herein by reference in their entirety. Such viruses are sometimes referred to as cytolytic or cytopathic viruses (or vectors), and, if they have such an affect on neoplastic cells, are referred to as oncolytic viruses (or vectors).
In one embodiment, the mutated adenoviral fiber protein of the invention is a fiber protein where at least one amino acid in the CD loop of a wild-type fiber protein of an _adenovirus from subgroup C, subgroup D,,, subgroup E, or. selected.viruses from subgroup F, (in particular those having the long fiber) have been mutated to reduce or substantially eliminate the ability of the fiber protein to bind to the cellular receptor known as the coxsackievirus-adenovirus receptor (CAR) to which the wild-type fiber of these subgroups, as well as subgroup A, bind. These subgroups are standard taxonomic designations known to those skilled in the art. Subgroup A includes adenovirus serotypes 12, 18, and 31. Subgroup C includes adenovirus serotypes 1, 2, 5, and 6. Subgroup D includes adenovirus serotype 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, and 42-49. Subgroup E
includes adenovirus serotype 4. Subgroup F includes adenovirus serotypes 40 and 41. These latter two serotypes have both a long and a short fiber protein. Only the long fiber protein binds to CAR. The -- ~ - preferred adenovirus serotype of the invention is. adenovirus .serotype 5~.
The reduction or elimination of the ability of the mutated adenovirus fiber protein to bind CAR as compared to the corresponding wild-type fiber protein is measured by comparing the transduction efficiency (gene transfer and expression of a marker gene) of an adenovirus particle containing the mutated fiber protein compared to an adenovirus particle containing the wild-type fiber protein for cells having CAR. As used herein, the term "substantially eliminate" refers to a transduction efficiency less than about 11% of the efficiency of the wild-type fiber containing virus on Hela cells using the transient transfection/infection system described in Example 1. Preferably, the efficiency is less than about 9%. Most preferably, the efficiency is less than about 8%. As used herein, the phrase "reduce" or "reduction" refers to a change in the efficiency of transduction by the adenovirus containing the mutated fiber as compared to the adenovirus containing the wild-type fiber to a level of about 75% or less of the wild-type on Hela cells using the transient SUBSTITUTE SHEET (RULE 26) transfection/infection system described in Example 1. Preferably, the change in efficiency is to a level of about 65% or less than wild-type. Most preferably, it is about 55% or less.
The fiber proteins of the invention are modified by chemical and biological techniques known to those skilled in the art. Such techniques permit the mutation of at~ least one amino acid in the CD loop of the wild-type fiber protein to change the ability of the protein to bind to CAR. As used herein, the term "mutate" or "mutation" or similar terms refers to the deletion or change of at least one amino acid in this part of the protein. The amino acid can be changed by substitution or by modification in a way that derivatizes the amino acid.
As mentioned above, the preferred fiber protein of the invention is a mutated -.adenovirus _ _ _ _ .. . . . ..serotype 5_.fiber protein. The . amino acid sequence ,of the , wild-type. protein is shown in SEQ ID N0:2. The CD loop in the wild-type adenovirus 5 protein extends from the amino acid at position 441 to the amino acid at position 453. Preferably, the amino acid at position 441 and/or the amino acid at position 442 of the wild-type fiber protein is mutated.
Such mutation may involve a deletion of the amino acid at either or both of positions 441 and 442 (SEQ ID NOS:6, 10, 12, 13). Alternatively, substitution at either or both of these positions may be made. In a particularly preferred embodiment, the amino acid at position 441 of the wild-type fiber protein is changed from valine to alanine. In another particularly preferred embodiment, the amino acid at position 442 of the wild-type fiber protein is changed from lysine to alanine. Most preferably, the amino acid at position 441 of the wild-';type fiber protein'is changed from valine to alanine, and the amino acid~at position~442 of-the . .=
wild-type fiber protein is changed from lysine to alanine (SEQ ID N0:14).
The present inventors have also discovered that certain mutations in other parts of the wild-type adenovirus 5 fiber protein reduce or substantially eliminate the ability of an adenoviral particle with the mutated fiber to bind to CAR. In a preferred embodiment, the mutations are at one or more of amino acid positions 408, 409, 460, 509, 510, 538, and 539 of the wild-type protein. In one particularly preferred embodiment, the fiber protein is mutated at amino acid positions 408 and 409, preferably by substituting glutamic acid for serine at position 408 and substituting alanine for proline at position 409 (SEQ ID
N0:4). In another preferred embodiment, the fiber protein is mutated at amino acid position 460 of the wild-type fiber protein, most preferably by substituting glutamic acid for arginine (SEQ ID
N0:16). In another preferred embodiment, the fiber protein is mutated at at least one of amino acid positions 509 and 510 of the wild-type fiber protein, preferably by deleting the 5.
SUBSTITUTE SHEET (RULE 26) amino acids at both positions (SEQ ID N0:18). In another preferred embodiment, the fiber protein is mutated at at least one of amino acid positions 538 and 539 of the wild-type fiber protein, preferably by deleting the amino acids at both positions (SEQ ID
N0:20).
Any or all of these mutations may be combined with mutations in the CD loop of adenovirus 5. In a preferred embodiment, the mutated fiber protein of the invention comprises at least one mutation at amino acid positions 441 and 442 of the wild-type fiber protein plus a mutation at one or more of amino acid positions 408, 409, 460, 509, 510, 538, and 539 of the wild-type fiber protein. For example, SEQ ID N0:8.
In an alternative, preferred embodiment, the mutated adenoviral fiber protein of the invention is a fiber protein where at least one amino acid in the AB loop of a wild-type fiber protein of an adenovirus from subgroup C, subgroup D, subgroup E, or selected viruses from .
subgroup F (in particular those having a long fiber), have been mutated to reduce or substantially eliminate the ability of the fiber protein to bind to CAR. In this embodiment, the preferred fiber protein of the invention is a mutated adenovirus serotype 5 fiber protein.
More preferably, the mutated adenovirus serotype 5 fiber protein contains mutations at amino acid positions 408 and/or 409 of the wild-type fiber protein.
Preferably, the mutations are at both positions. As mentioned previously, such mutations may be deletions, substitutions, or a modification in a way that derivitizes the amino acid. The same type of mutation need not be made at each position. In one preferred version of this preferred embodiment, glutamic acid is substituted for serine at position 408. In an alternative preferred version-of this preferred ernbodiment,~»alanine is substituted.~for proline at<.position.,;~
409. Most preferably, glutamic acid is substituted for serine at position 408, and alanine is substituted for proline at position 409 (SEQ ID N0:4).
The invention also comprises polynucleotides that encode the proteins of the invention. As used herein, the term "polynucleotide" means a nucleic acid molecule, such as DNA or RNA, that encodes a polynucleotide. The molecule may include regulatory sequences. Preferably, the polynucleotide is DNA. Such polynucleotides are prepared or obtained by techniques known by those skilled in the art in combination with the teachings contained therein. Examples of such polynucleotides are shown in SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19. The polynucleotides of the invention also include polynucleotides that differ in certain bases but still encode the proteins of the invention due to the redundancy of the genetic code.

SUBSTITUTE SHEET (RULE 26) The invention further comprises vectors including the polynucleotides of the invention. Such vectors include partial or complete adenoviral genomes and plasmids. Such vectors are constructed by techniques known to those skilled in the art.
One of the principal uses of such vectors is the production of adenoviral packaging cells. The packaging cells of the invention are cells that provide complementing functions to the functions provided by the genes in the adenovirus genome that are to be packaged into the adenovirus particle. The production of such particles require that the genome be replicated and that those proteins necessary for assembling an infectious virus be produced. The particles may also require certain proteins necessary for the maturation of the viral particle.
Such proteins may be provided by the vector or by the packaging cell.
.. The packaging cells of the invention. may contain'theepolynucleotide encoding the mutated fiber protein. Such polynucleotide may be transfected into the cell, preferably as part of a plasmid, or it may be infected into the cell with a viral vector. It may be stably incorporated into the genome of the cell, thus providing for a stable cell line. Alternatively, it may be unincorporated into the genome, in which case a transient complementing cell will be provided.
The adenovirus genome to be packaged is transferred into the complementing cell by techniques known to those skilled in the art. These techniques include transfection or infection with another virus. The polynucleotide encoding the mutated fiber protein may be in this genome instead of in the packaging cell.
"In certain cases, it may be desirable, when the polynucleotide~ encoding the mutated fiber is in the genome to be packaged, for the packaging cell to also encode a fiber protein.
Such protein may assist in the maturation and packaging of an infectious particle. Such protein may be a wild-type fiber protein or one modified so as to be unable to attach to the penton base protein.
The packaging cells are cultured under conditions that permit the production of the desired viral particle. The viral particles are recovered by standard techniques.
A preferred way of making the adenoviral particles of the invention is as follows. The polynucleotide encoding the mutated fiber protein is made using standard techniques in an adenoviral shuttle plasmid. This plasmid contains the right end of the virus, in particular from the end of the E3 region through the right ITR. It also contains a recombinase site, such as a lox site. This plasmid is co-transfected into a complementing cell line along with a helper plasmid, which contains the remaining portion of the adenovirus genome, except for SUBSTITUTE SHEET (RULE 26) the E1 region and sometimes also the E2a region. A third plasmid, which is an expression plasmid containing a gene encoding a recombinase such as Cre, is also transfected into the complementing cell. The complementing cell is preferably a 293 cell, which contains the adenoviral E1 genes, or an AE1-2a cell {Gorziglia, Kadan, et al. 1996}, which contains the adenoviral EI and E2a genes. Most preferably, the complementing cell is a 633 cell {Von Seggern, Huang, et al. 2000}, which stably expresses the adenovirus serotype 5 wild-type fiber protein, and was derived from the AE1-2a cell line.
The transfected complementing cells are maintained under standard cell culture conditions. The adenoviral plasmids recombine to form the adenoviral genome that is packaged. The particles are infectious, but replication deficient because their genome is mis~zng at. least, the E1._ genes.. The, particles contain both wild-type and mutated,.fiber .. .
proteins. They are recovered from the crude viral lysate and are purified by standard techniques.
The recovered particles are preferably used to infect 293 or AE1-2a cells.
This permits the recovery of particles whose capsids contain only the desired mutated fiber. This two-step procedure provides high titer batches of the adenoviral particles of the invention.
The adenoviral particles may be replication competent or replication incompetent. In a preferred embodiment of the invention, the particles selectively replicate in certain predetermined target tissue but are replication incompetent in other cells and tissues. In a particularly preferred embodiment of the invention, the adenoviral particles replicate in ~~ °abnormally proliferating- tissue, such as solid tumors and other~..neoplasms. -Sueh..,replication , conditional adenoviral particles and vectors may be produced by techniques known to those skilled in the art, such as those disclosed in the above-referenced U.S.
Patent Nos. 5,998,205 and 5,801,029. These particles and vectors may be produced in adenoviral packaging cells as disclosed above. The preferred packaging cells are those that have been designed to limit homologous recombination that could lead to wild-type adenoviral particles.
Such cells are disclosed in U.S. Patent Nos. 5,994,128, issued November 30, 1999 to Fallaux, et al., and 6,033,908, issued March 7, 2000 to Bout, et al. The packaging cell known as PER.C6, which is disclosed in these patents, is particularly preferred.
Preferably, the modified fiber palynucleotide also includes sequences that encode a targeting ligand. Accordingly, such sequences are transfected into the complementing cell by the shuttle plasmid.

SUBSTITUTE SHEET (RULE 26) Alternatively, the targeting Iigand sequences may be included in the penton or hexon proteins. In such cases, they would be in the helper plasmid.
The packaged adenoviral genome may also contain a heterologous polynucleotide.
This polynucleotide is usually included in the deleted E1 region of the helper plasmid.
Alternatively, the polynucleotide may be in the E3 region, in which case it is included in the shuttle plasmid.
The adenovirus particles of the invention include the mutated fiber proteins.
Such particles may include different types of the mutated fibers of the invention.
They may also include wild-type fibers along with the mutated fibers.
The adenoviral particles of the invention preferably further comprise a targeting ligand included in a capsid protein of the particle. The ligand may be included in any"of the, capsid proteins, such as fiber, hexon, or penton. Preferably, the ligand is included in a fiber protein, which is preferably a mutated fiber protein of the invention. Most preferably, the targeting ligand is included within the HI loop of the fiber protein. Any ligand that can fit in the HI loop and still provide a functional virus may be used. Such ligands may be as long as 80-90 amino acids. Such ligands are added by techniques known in the art, such as those disclosed in PCT application PCT/LJS99/02549, published as W099/39734 on August 12, 1999, and Example 12 of U.S. patent application number 091482,682, filed January 14, 2000, the disclosures of both of which are hereby incorporated herein by reference.
A targeting ligand may be any chemical moiety that preferentially directs the adenovira.l particle to ~-a desired~~cell~~type:~ Thev..categories of such ligands include peptides, ~--polypeptides, single chain antibodies, and multimeric proteins. Specific ligands include the TNF superfamily of ligands which include tumor necrosis factors (or TNF's) such as, for example, TNF-alpha and TNF-beta, lymphotoxins (LT), such as LT-a and LT-j3, Fas ligand which binds to Fas antigen; CD40 ligand, which binds to the CD40 receptor of B-lymphocytes; CD30 ligand, which binds to the CD30 receptor of neoplastic cells of Hodgkin's lymphoma; CD27 ligand, NGF ligand, and OX-40 ligand; transferrin, which binds to the transferrin receptor located on tumor cells, activated T-cells, and neural tissue cells;
ApoB, which binds to the LDL receptor of liver cells; alpha-2-macroglobulin, which binds to the LRP receptor of liver cells; alpha-1 acid glycoprotein, which binds to the asialoglycoprotein receptor of liver; mannose-containing peptides, which bind to the mannose receptor of macrophages; sialyl-Lewis-X antigen-containing peptides, which bind to the ELAM-1 receptor of activated endothelial cells; CD34 ligand, which binds to the CD34 SUBSTITUTE SHEET (RULE 26) ligand, which binds to the CD34 receptor of hematopoietic progenitor cells;
ICAM-1, which binds to the LFA-1 (CDllb/CD18) receptor of lymphocytes, or to the Mac-1 (CDlla/CD18) receptor of macrophages; M-CSF, which binds to the c-fms receptor of spleen and bone marrow macrophages; circumsporozoite protein, which binds to hepatic Plasmodium falciparum receptor of liver cells; VLA-4, which binds to the VCAM-1 receptor of activated endothelial cells; HIV gp 120 and Class II MHC antigen, which bind to the CD4 receptor of T-helper cells; the LDL receptor binding region of the apolipoprotein E (ApoE) molecule;
colony stimulating factor, or CSF, which binds to the CSF receptor; insulin-like growth factors, such as IGF-I and IGF-II, which bind to the IGF-1 and IGF-II
receptors, respectively;
Interleukins 1 through 14, which bind to the Interleukin 1 through 14 receptors, respectively;
the Fv.antigen-binding domain of an immunoglobulin; and cyclic RGD peptide.
Cyclic RGD
(cRGD) is preferred. As used herein, the term "cyclic RGD" (or cRGD) refers to any amino acid that binds to a integrins on the surface of cells and contains the sequence RGD (Arg-Gly-Asp). The sequence in SEQ ID N0:43 is particularly preferred.
In a preferred embodiment because the adenovirus fiber has a trimeric structure, the ligand also has a trimeric structure. In a more preferred embodiment, the ligand is selected from the TNF superfamily of ligands hereinabove described. Such ligands are trimeric and of similar size to the fiber head domain. Such ligands may be incorporated into the fiber protein using the techniques disclosed in U.S. Patent No. 5,756,086, issued May 26, 1998 to McClelland et al., the disclosure of which is incorporated herein by reference.
~Tlie adenovirus particle's mayfurther'include at leastwone°heterologous polynucleotide.
As used herein, the term "heterologous polynucleotide" means a polynucleotide derived from a biological source other than an adenovirus, which encodes a polypeptide when the adenovirus infects a cell. Such polynucleotides are included in the adenoviral genome within the particle and are added to that genome by techniques known in the art. Any heterologous polynucleotide of interest may be added. A preferred polynucleotide is one that encodes an immunostimulating protein, such as an interleukin, interferon, or colony stimulating factor.
Mammalian GM-CSF is preferred. Preferably, such GM-CSF is a primate GM-CSF;
most preferably, it is human GM-CSF. An alternative preferred polynucleotide encodes herpes simplex virus thymidine kinase (HSV-TK), which is useful as a safety switch as described in U.S. Patent Application No. 08/974,391, filed November 19, 1997, which published as PCT
Publication No. W0/9925860, the disclosure of which is incorporated herein by reference.
to SUBSTITUTE SHEET (RULE 26) The adenoviral particles of the invention are used to genetically engineer a cell to express a protein that it otherwise does not express or does not express in sufficient quantities. This is accomplished by infecting the desired cell with an adenoviral particle of the invention whose genome includes a desired heterologous polynucleotide.
This permits the expression of the heterologous polynucleotide in the cell. Preferably, the cell is a mammalian cell. More preferably, the mammalian cell is a primate cell. Most preferably, the primate cell is a human cell. The cell may be inside the body of the animal (ih vivo) or outside the body (i~c vitro).
Preferably, the adenoviral particle includes a targeting ligand as described above.
This permits the delivery of a gene to a desired cell type which is different from the cell type that. wild-type adenovirus_ particle infect or the. same as a wild-type ,particle, would infect but _ , allowing the infection in a selective manner, i.e., non-target cell types would not be infected.
Thus, the adenoviruses of the invention can be used to study cell transduction and gene expression in vitro or in various animal models. The latter case includes ex vivo techniques, in which cells are transduced ih vitro and then administered to the animal. They may also be used to conduct gene therapy on humans or other animals. Such gene therapy may be ex vivo or ih vivo. For in vivo gene therapy, the adenoviral particles of the invention in a pharmaceutically-acceptable carrier are delivered to a human in a therapeutically effective amount in order to prevent, treat, or ameliorate a disease or other medical condition in the human through the introduction of a heterologous gene that encodes a therapeutic I
- protein into=cells~ in~-»suchhuman. The adenoviruses are delivered at .a dose ranging from r.. '~
approximately 1 particle per kilogram of body weight to approximately 1014 particles per kilogram of body weight. Preferably, they are delivered at a dose of approximately 106 particles per kilogram of body weight to approximately 1013 particles per kilogram of body weight. Most preferably, the dose ranges from approximately 109 particles per kilogram of body weight to approximately 1012 particles per kilogram of body weight.
The adenoviral particles of the invention with the above-identified modifications in the AB loop, particularly those with modifications at amino acid positions 408 and 409 of the wild-type adenovirus serotype 5 fiber protein, and most particularly those where glutamic acid is substituted fox serine at position 408 and alanine is substituted for proline at position 409 (SEQ m N0:4), have additional desirable utilities. The inventors have unexpectedly discovered that such viral particles provide enhanced gene transfer to and expressionism hepatocytes in the liver of an animal as compared to adenoviral particles with the wild-type SUBSTITUTE SHEET (RULE 26) fiber protein. Therefore, the invention includes a method of enhancing adenoviral-mediated gene transfer to and expression in cells in the liver of an animal by administering adenoviruses having such AB loop modification in at least one of their fiber proteins to an animal under conditions where cells in the liver are transduced. The hepatocytes are the cells that are primarily transduced. Preferably, an adenovirus particle comprising a mutated adenovirus serotype 5 fiber protein, wherein glutamic acid is substituted for serine at amino acid position 408 and alanine is substituted for proline at amino acid position 409 (SEQ ID
N0:4), is used to deliver the heterologous gene.
Such adenoviral particles would be particularly useful for gene therapy where it is desired to express a heterologous gene in a patient's liver. This could be used, for example, in the treatment of diabetes, hemophilia, and . diseases related to increased cholesterol or triglyceride blood levels in a patient such as atherosclerosis. It would also include anti-angiogenesis treatment methods involving the delivery of one or more anti-angiogenic genes to the hepatocytes of a patient's liver.
Because of the enhanced gene transfer and expression, lower dose) of these viral particles would be able to be used. The dose for these types of particles would be approximately 1 particle per kilogram of body weight to approximately 1013 particles per kilogram of body weight. Preferably, the dose would be approximately 105 particles per kilogram of body weight to approximately 1012 particles per kilogram of body weight. Most preferably, the dose ranges from approximately 10g particles per kilogram of body weight to approximately~I011yarticles per°kilograrnnfbodywweight.
Such particles are delivered by routes of administration known to those skilled in the art. One such route is intravenous injection. An alternative route is intraparenchymal injection. The particles may also be delivered by injection into the hepatic artery, portal vein, or bile duct.
Another use of the particles with the AB loop mutations would be as a basis for further modification of the particles, wherein the RGD amino acid sequence in the penton protein is deleted, modified, or substituted. Techniques for such modifications are disclosed in U.S. Patent Nos. 5,559,099, issued September 24, 1996 to Wickham, et al., 5,712,136, issued January 27, 1998 to Wickham, et al., and 5,731,190, issued March 24, 1998 to Wickham, et al., the disclosures of which are incorporated herein by reference. This would prevent the particles from binding to cell surface integrins and being taken into the cell.

SUBSTITUTE SHEET (RULE 26) EXAMPLES
Example 1 Adenovirus Type 5 Viral Particles Pseudotyped With Mutagenized Fiber Proteins Show Diminished Infectivity of Coxsackie B-Adenovirus Receptor-Bearing Cells INTRODUCTION
The great interest in human adenovirus type 5 (Ad5) as a gene delivery platform is due in part to its ability to efficiently infect many cell types. Its wide tropism is mediated by a primary interaction between the Ad5 capsid protein, fiber, and its high-affinity cellular receptor, the coxsackie adenovirus receptor (CAR). Fiber is a homotrimeric protein present twelwe~~tirizes 'on the viral capsid. It has three domains: an N-terminal tail that interacts with the penton base in the viral capsid, a rod-like shaft containing 22 copies of a 15 amino-acid beta sheet structure, and a globular knob domain. It is the knob domain that mediates binding to CAR during cell attachment. After the initial binding event, a second, low-affinity interaction takes place between the penton base and oc" integrins on the cell surface. This step ..
is required for virus internalization and subsequent gene transfer.
There are many cases where it is desirable to deliver therapeutic genes to a subset of cell types. For this reason, there has been much effort to specifically target Ad5 vectors. This capability involves the detargeting away from its natural receptor and the simultaneous retargeting of the viral tropism toward a given cell type. The resulting vector would represent an irriportamt step in the development of this gene therapy plat~oim,~ both from an efficacy and a safety standpoint. Reducing the undesired virus-tissue interactions and increasing the intended interaction would allow lower viral doses to be used and thereby potentially minimize the associated toxic side effects and host immune response.
Several strategies have been used to alter the receptor tropism and binding specificity of adenoviral vectors. These strategies include replacing the fiber knob domain with a knob from another Ad serotype with a different receptor specificity { Stevenson, Rollence, et al.
1995 } { Krasnykh, Mikheeva, et al. 1996 } { Stevenson, Rollence, et al. 1997 }, the insertion of peptides onto the C-terminus of fiber {Wickham, Granados, et al.
1990}{Michael, Hong, et al. 1995 } { Wickham, Tzeng, et al. 1997 } or the exposed HI loop { Krasnykh, Dmitriev, et al.
1998} and the use of bifunctional antibodies {Wickham, Segal, et al. 1996}.
The results of these efforts have been an expansion of viral tropism, which is suitable for some gene therapy applications such as vascular gene therapy where the aim is to improve the gene transfer SUBSTITUTE SHEET (RULE 26) efficiency of Ad vectors that are delivered locally. However, to specifically transduce certain cell types with systemically-delivered adenoviral vectors, it will be necessary to ablate the natural receptor tropism in combination with the introduction of a high affinity targeting ligand.
The analysis of multiple fiber mutations in the context of a viral particle remains a tedious process that involves the time-consuming incorporation of modified fiber genes into the adenoviral genomic DNA. Furthermore, the incorporation of mutafied fiber genes into the Ad genome may affect the efficient growth and propagation of Ad. As a result, the generation and evaluation of adenoviral vectors containing mutated fiber proteins may require alternative means of growing the vectors that will allow for the efficient production of high titer viral.stocks. .. ... _._ .... . . _ . . _. . .. . . ..~ _ . . _ We have developed a novel system to rapidly analyze modified fiber proteins for desired tropism in the context of the viral particle. This system is based on the ability to pseudotype a fiberless Ad5 mutant with fiber proteins expressed transiently from an episomal plasmid (Fig. 1). The fiber-deleted Ad vector is AdS.(3gal..aF, which is an E1-E3- and fiber-gene deleted adenovirus that expresses cytoplasmic (3-galactosidase under the control of the SV40 promoter {Von Seggern, Chiu, et al. 1999} (Fig. 1A). The modified fiber proteins for pseudotyping are produced from expression plasmid constructs designed for high levels of fiber protein expression { Von Seggern, Huang, et al. 2000 } . The primary advantage of this system is that modified fiber proteins can be quickly incorporated into virions and functionally analyzed"im' their 'most relevant context for their effectwon CAR° interaction and ~ . °
subsequent gene transfer and expression. We used this system to analyze a panel of fiber mutants for their ability to mediate adenoviral gene transfer to Hela cells, a CAR-expressing cell line. We have shown that the transient transfection/infection system can efficiently pseudotype a fiberless viral capsid with levels of fiber protein indistinguishable from those seen on wildtype virions. We used this system to identify multiple fiber gene mutations that significantly reduce the, ability of adenovirus to transduce cells and achieve gene transfer.
MATERIALS AND METHODS
Plasmids and fiber glene mutaffenesis. The Ad5 fiber cDNA has been cloned into pcDNA3.l to generate pDV60, as previously described {Von Seggern, Huang, et al. 2000}.
Briefly, pDV60 contains the CMV promoter, the first Ad5 tripartite leader exon (TPL), the natural first intron and the fused second and third TPL exons upstream of the Ad5 fiber gene.

SUBSTITUTE SHEET (RULE 26) All amino acid changes were incorporated into the fiber cDNA using the pDV60 plasmid as the template. Individual amino acid residues in pDV60 were mutagenized using the QuickChange Site-Directed Mutagenesis system (Stratagene, La Jolla CA). The oligonucleotide primers used for the incorporation of amino acid changes are listed in Table 1 for each single or double amino acid modification. The thermal cycler protocol was 95°C for 30 sec, followed by 18 cycles of 95°C for 30 sec, 55°C for 1 min, and 68°C for 20 min.
The entire knob domain of the Ad5 fiber was deleted from amino acids 404 to 581.
For restoration of trimerization of the fiber tail and shaft, a 31 amino acid peptide derived from the GCN4 leucine zipper {Harbury, Zhang, et al. 1993 } was fused immediately after the fiber TLWT sequence at the fiber shaft-head junction using PCR gene overlap extension {Norton, Cai, et al. 1990}. This, reaction fused,the.Ad5 fiber,tail and shaft regions (amino acids 1 to 403) to the GCN4 isoleucine 31 amino acid peptide to form the KO11 mutant and was cloned into pDV60 to create pDK011. For all fiber mutations, the nucleotide sequence of the cloned insert was determined and in each case a clone having the expected sequence was selected. The pDV55 contre~l plasmid is similar to pDV60, except that it lacks the fiber gene { Von Seggern, Huang, et al. 2000 } .
Viruses. AdS.(3gal.wt is a first generation E1-, E3-deleted adenovirus containing a lacZ reporter cassette in the E1 region {Von Seggern, Chiu, et al. 1999}.
AdS.(3gal.OF is identical to Ad5.~3gal.wt except that the fiber gene is deleted {Von Seggern, Chiu, et al.
1999 } .
Cells. Human 293T cells'wer'e''obtained frorri ATCC'°(CRL 11268) and were cultured in the DMEM containing 10% FBS. The 293T cells stably express the SV40 large T
antigen that allows for the amplification of plasmids from the SV40 origin of replication. The 633 cells stably express the Ad5 fiber protein {Von Seggern, Huang, et al. 2000}
and are derived from AE1-2a, a cell line that complements Ela- and E2a-deleted adenoviral vectors { Gorziglia, Kadan, et al. 1996 } . 633 cells were grown in Richter's CM (Life Technologies #C-2671) and 10% FBS. Hela cells (ATCC CCL-2) were grown in Dulbecco's modified Eagle's media supplemented with 10% FBS. For a further description of the preparation of AEl-2a cells, also known as S8 cells, and the preparation of 633 cells, see Example 6F and 6G of U.S. Patent Application number 09/482,682, filed January 14, 2000, which disclosure is incorporated herein by reference.
Transient Transfection/Infection. Mutated fiber proteins were incorporated into adenoviral particles using the transient transfection/infection system. For each virus SUBSTITUTE SHEET (RULE 26) preparation using the transient transfection/infection system, four 15 cm dishes of 70%
confluent 293T cells were used. For transfections, IOOug of each fiber expression plasmid DNA listed in Table 2, 400u1 lipofectamine (Life Technologies, Rockville, MD) and 3.6m1 Opti-MEM 1 media (Life Technologies, Rockville, MD) were combined in a conical 250m1 sterile bottle. At the end of a 30 minute room temperature incubation, 60m1 Opti-MEM 1 media was added. A 16m1 aliquot of this transfection mix was added to each plate and incubated at 37°C, 5% COZ for 5 hours. The transfection media was then aspirated and 20 ml of complete DMEM media was added. The dishes were then incubated at 37°C, 5% COZ for 24 hours to allow expression of the fiber protein.
The transfected 293T cells were then infected with AdS.(3Gal.OF/F+ virus at a particle per cell ratio of .350. The AdS.(3Gal.~F/F+ virus is an E1, E3, fiber-deleted.Ad5 vector .{.Von _ Seggern, Chiu, et al. 1999 } that was propagated in the fiber-complementing cell line, 633, such that the capsid contains wildtype Ad5 fiber protein {Von Seggern, Huang, et al. 2000}.
The growth media was aspirated and 2.5m1 of infection media (DMEM and 2% FBS) containing AdS:(3Gal.OF/F+ was added and slowly rocked at 37°C, 5% COZ
for 2 hours.
Twenty ml of growth media was then added (DMEM and 10% FBS) and the plates were incubated at 37°C, 5% COZ overnight. The media was replaced the next day and the incubation was continued until complete cytopathic effect (CPE) was observed, typically in about 3 to 4 days. The transfected/infected 293T cells were harvested after complete CPE by gently dislodging the cells, pelleting by centrifugation, and resuspending in lml phosphate ~buffered° -salime. A ci ude viral lysate (CVL) was prepaxed' liy five freeze 'thfi.v~ cycles ' to disrupt the cells and release the virus. The virus was purified by CsCI
gradient centrifugation using standard procedures. The virus particle titer was determined spectrophotometrically as described {Mittereder, March, et al. 1996}. Yields of AdS.(3Gal.~F virus pseudotyped with modified fiber protein typically ranged from 1011 to 1012 particles.
Western immunoblot analysis. The expression and incorporation of each fiber protein onto adenoviral particles was verified by denaturing sodium dodecyl-sulfate (SDS) 4 to 12%
polyacrylamide gel electrophoresis (PAGE) and Western immunoblot analysis. An aliquot of each adenoviral vector preparation corresponding to 5.0x109 particles per lane was analyzed.
The proteins were transferred to a nitrocellulose membrane with a minitransblot apparatus (Novex Inc.) for 90 minutes at 30V. The membrane was blocked for at least 1 hour at room temperature in lOmM Tris, pH7.4 containing 150mM NaCl, 2mM EDTA, 0.04% Tween-20, and 5% dried milk. The blocked membrane was incubated for 1 hour with a 1:1000 dilution SUBSTITUTE SHEET (RULE 26) of a primary rabbit anti-Ad5 fiber polyclonal antiserum. The membrane was then developed with a 1:5000 dilution of the secondary donkey anti-rabbit IgG horseradish peroxidase-conjugated antibody (Amersham Lifesciences, Arlington Heights, IL) using an enhanced chemiluminescense system (Amersham Lifesciences). The membrane was exposed to film for approximately 1 to 20 seconds. The membrane was then used to reprobe for detection of the adenoviral penton protein to ensure equivalent loading of viral particles.
Briefly, the membrane was incubated for 1 hour with a 1:5000 dilution of the primary rabbit anti-Ad5 penton polyclonal antiserum. The membrane was then re-developed with a 1:5000 dilution of the secondary goat anti-rabbit IgG horseradish peroxidase-conjugated antibody as described above.
' Production of anti-Ad5- fiber and anti-Ad5 penton-specific antiserum:wBothwof-the-rabbit primary antibodies used in the anti-fiber and anti-penton Western immunoblot analysis were generated by immunizations of New Zealand White rabbits (Loftstrand Labs, Ltd., Gaithersburg, MD). The Ad5 fiber and penton proteins were expressed using the baculoviral expression system. The purified Ad5 fiber protein and partially purified.penton base proteins were used for immunizations according to standard protocols. The antiserum obtained was tested for immunoreactivity against the Ad5 fiber and penton proteins by Western immunoblot analysis.
Adenoviral transduction. Hela cells were infected with the adenoviral vectors containing mutated fiber proteins to evaluate the effects of fiber amino acid, mutations, on CAR interaction and subsequent gene expression. Monolayers of HeLa cells in 12 well dishes were infected with 1000 particles per cell fox 2 hours at 37°C in a total volume of 0.35 ml of the DMEM containing 2% FBS. The infection medium was then aspirated from the monolayers and lml of complete DMEM containing 10% FBS was added per well. The cells were incubated for an additional 24 hours to allow for (3-galactosidase expression.
~i-aalactosidase expression anah!sis. The expression of (3-galactosidase encoded by the adenoviral vectors in the ,infected cells was measured by a chemiluminescence reporter assay and by histochemical staining with a chromogenic substrate. The relative levels of (3-galactosidase activity were determined using the Galacto-Light chemiluminescence reporter assay system (Tropix, Bedford, MA). Briefly, the cell monolayers were washed with PBS and processed according to the manufacturer's protocol. The cell homogenate was transferred to a microfuge tube and centrifuged to remove cellular debris. Total protein concentration was SUBSTITUTE SHEET (RULE 26) determined using the bicinchoninic acid (BCA) protein assay (Pierce, Inc., Rockford, IL) with bovine serum albumin as the assay standard. An aliquot of each sample was then incubated with the Tropix ~i-galactosidase substrate for 45 minutes in a 96 well plate. A
luminometer was used to determine the relative light units (RLU) emitted per sample and then normalized for the amount of total protein in each sample (RLU/ug total protein). For the histochemical staining procedure, the cell monolayers were fixed with 0.5°70 glutaraldehyde in PBS, and then were incubated with a mixture of 1 mg of 5-bromo-4-chloro-3-indolyl-~i-D-galactoside (X-gal) per ml, 5mM potassium ferrocyanide, 5mM
potassium ferricyanide and 2 mM MgCl2 in 0.5 ml of PBS. The monolayers were washed with PBS and the blue cells were visualized by light microscopy with a Zeiss ID03 microscope.
RESULTS
Transient transfection/infection system. To rapidly evaluate a panel of potential CAR-binding fiber mutants in the context of viral particles, we have developed a transient transfectionlinfection system. This system, which is based on pseudotyping a fiberless virus ,. .
with the mutant fiber proteins, consists of two components. The first is an E1, E3, fiber-deleted adenovirus, AdS.(3gal.OF {Von Seggern, Chiu, et al. 1999} (Fig. 1A).
This virus, when grown on a non fiber-complementing cell line such as 293T, yields viral particles lacking fiber protein. For purposes here, these fiberless virions are designated AdS.[3gal.~F/F-. If AdS.(3gal.~F is produced on the fiber-complementing cell line 633 {Von Seggern, Huang, et al. 2000}, the virions contain a full corripTement'bf wildtype fiber protein on the surface and is referred to as AdS.(3gal.OF/F+. The second component of the system is an expression plasmid that supplies fiber protein to the assembling virus in trays. This plasmid, pDV60, is designed to express high levels of fiber protein {Von Seggern, Huang, et al. 2000 } .
The transient transfection/infection system is shown schematically in Fig. 1B.
Transfection of 293T cells .by the pDV60-based, fiber-expression, plasmid results in high levels of fiber production in the cells. Twenty-four hours later, the cells are infected with AdS.[3gal.~F/F+ that has been previously pseudotyped with wildtype fiber by growth in 633 cells. Approximately three days later, the infected cells are collected and viral particles, now pseudotyped with the fiber protein supplied in tYahs by the fiber-expression plasmid, are purified. In this way, any plasmid-encoded fiber proteins that are capable of trimerization and incorporating into the viral particles will complement AdS.(3gal.~F.
AdS.(3gal.OF that is SUBSTITUTE SHEET (RULE 26) pseudotyped either by.growth in 633 cells or by transient transfection with a fiber expression plasmid is designated AdS.(3gal.AF/F+. The function of these modified fiber proteins in the context of a viral particle can then be tested for their ability to mediate fiber-dependent Ad infection and gene transfer.
To compare the Ievel of fiber protein incorporated onto AdS.(3gal.dF/F+ viral particles generated by this system with the levels in AdS. (3gal.wt, Western immunoblot analysis was performed. Equal particle numbers of AdS.(3gal.OF/F-, AdS.(3gal.~F/F+
pseudotyped by pDV60-encoded fiber protein, and AdS.(3gal.wt were evaluated for fiber and penton protein levels. As reported previously {Von Seggern, Chiu, et al. 1999}, the AdS.(3gal.OF/F- virions lacked any detectable fiber protein, and AdS.(3gal.wt contained expected levels of the 62kDa fiber~~protein. Importaritly~ the level of pDV60-encoded fiber protein incorporated into the AdS.(3gal.OF/F+ pseudotyped virions using the transient transfection/infection system was equivalent to the level of fiber protein in the AdS. (3gal.wt particles. The equivalent loading of viral particles was demonstrated by detection of the 68kDa penton monomer for each vector.
These results indicate that expression of fiber protein in traps from this expression plasmid can complement AdS.(3gal.~F and can result in a level of fiber protein on the capsid that is indistinguishable from that of an adenovirus containing fiber within its genome.
Fiber mutation anal. The transient transfection/infection system was then used to evaluate a series of mutations in the fiber knob for their effect on CAR-mediated gene transfer of Ad5 particles. A panel of expression plasmids encoding fourteen mutant fiber proteins was constructed (Table 2). As controls, the wildtype fiber (pDV60) and a null construct (pDV55) were used {Von Seggern, Huang, et al. 2000}. These plasmids were transfected into 293T cells, followed by infection with AdS.(3gal.OF/F+. The resulting virions obtained from this procedure were thus pseudotyped with the plasmid-encoded fibers. The expression and assembly of each fiber protein into the adenoviral capsid was examined by Western immunoblot analysis of the CsCI-purified virus stocks. The relative levels of fiber protein on' the capsid were compared with the amount of penton protein to control for equal loading of viral particles in each lane. The fiber proteins encoded by most mutants were sufficiently expressed, trimerized and incorporated into the AdS.(3gal.~F
viral particles and the 62kDa fiber monomer was detected in this analysis. Analysis of the KO11 mutant displayed the expected protein of approximately 48kDa although this truncated fiber protein was not incorporated to the same level as wild-type fiber. These results demonstrate that the mutations introduced into the majority of these fiber genes did not impair their ability to be SUBSTITUTE SHEET (RULE 26) expressed, trimerized and incorporated into viral particles at levels indistinguishable from wild-type fiber. However, mutants K02 (SEQ 117 N0:6), KO1+2 (SEQ ID N0:8), K02a (SEQ ID NO:10), and KO11 showed lower levels of incorporated fiber protein although KO11 may have a reduced immunoreactivity with an antiserum that was generated against the full-length wildtype Ad5 fiber protein. Analysis of the relative expression level and trimerization ability of these mutants on non-denaturing polyacrylamide gels showed lower levels of fiber monomer and trimer, indicative of deficiencies in the steady-state levels of these mutant proteins. Except for KO11, which is a deletion of the entire knob, all of these mutants have a mutation at V441 in common.
Having demonstrated efficient expression in traps and virion incorporation of most of these mutant fiber proteins, we next evaluated .the affects of these mutations on functional CAR-binding properties. We did this by comparing the transduction efficiency on Hela cells of virions pseudotyped with mutant fiber protein and those pseudotyped with wildtype fiber protein. Transduction efficiency was measured in two ways. A chemiluminescence reporter assay was used to measure the level of adenoviral-encoded (3-galactosidase activity and the values (RLU/ug total cellular protein) from one representative experiment are shown in Table 2. A total of five to six separate transductions were performed and the mean 0 (3-galactosidase activity values (RLU/ug total cellular protein) were calculated for each adenoviral vector containing the individual fiber mutants. These values were then normalized to the (3-galactosidase activity chemiluminescence values obtained with the wildtype fiber to obtain the relative activity of'each mutant compared to wildtype fiber. 'The F-, fiberless vector displayed the most significant, 1000-fold reduction in transduction with only 0.1 % of wt activity demonstrating the need for fiber in the efficient transduction of HeLa cells. The KO1 mutation displayed approximately a 70-fold reduction resulting in only 1.4% wt (3-galactosidase activity levels. The K02 (SEQ ID N0:6) mutation resulted in an approximately 167-fold reduction with 0.6% wt activity. The K02a (SEQ ID NO:10), K02b (SEQ
ID
N0:12), and K02c._(SEQ ID, NO:14) constructs were designed to identify the amino acid mutation responsible for the significant reduction in CAR interaction. In this comparison, it was revealed that the deletion of amino acid V441 reduced CAR interaction greatest as this single deletion in K02a (SEQ ID NO:10) resulted in the 167-fold reduction of wt activity and the deletion of K442 had no further effect. The most potent mutation was found with combining the KO1 (SEQ ID N0:4) and K02 (SEQ ID N0:6) mutations in the KO1+2 (SEQ
ID N0:8) construct as this combination resulted in a 1000-fold reduction with only 0.1% wt SUBSTITUTE SHEET (RULE 26) levels found. A dramatic reduction in transduction efficiency was also found for the KO11 mutant with a 125-fold reduction of (3-galactosidase activity when the entire knob domain was deleted. Significant decreases were also observed for K03 (SEQ ID N0:16), K04 (SEQ
ID N0:18), and KO5 (SEQ ID N0:20) although not as dramatic. Little to no effect on fiber-mediated transduction and gene expression was noted for K04+5, K08, K09 and KO10. The average percent wild-type (3-galactosidase activity for each pseudotyped adenoviral vector is shown graphically in Figure 2.
We also analyzed the transduction efficiency of the pseudotyped AdS.(3gal.OF/F+ by measuring the percentage of cells that were positive for the lacZ reporter gene. This was done by staining the transduced cell monolayers with X-gal. For all mutants, the histochemical data was consistent with the chemiluminescence data. At 1000 ~paxt~icles per cell, Hela cells infected with AdS. [3gal.OF/F+ pseudotyped with pDV60 showed a high percentage of blue, X-gal positive cells, while AdS. (3gal.~F/F- pseudotyped with pDV55 demonstrated very few if any blue cells. The mutants KO1 (SEQ ID N0:4), K02a (SEQ 1n NO:10), and KO2 (SEQ
ID N0:6) and K02c (SEQ ID N0:14), which showed dramatically lower ~3-galactosidase;
activity (Table 2 and Fig. 2), also showed extremely low numbers of blue cells as expected.
K04 showed an intermediate reduction in (3-galactosidase activity and in the number of X-gal stained positive cells while KO10 had little effect on transduction efficiency by either measure (Fig. 2).
DIS CUS SIGN
One approach to selective cell transduction is to manipulate the fiber protein to redirect the receptor specificity to a particular cell type. There are two requirements for such a targeted adenovirus gene therapy vector. One is ablation of the natural tropism and the second is the introduction of a novel tropism to desired target cell types. We developed a novel system to analyze mutant fiber proteins in the context of the viral particle. Using this system, we identified a number of mutant fiber proteins that retained the ability to incorporate into viral particles but demonstrated a reduction in fiber-mediated gene transfer due to a diminished interaction with CAR. The most dramatic mutations were KO11 0404-581) and the mutations localized to the fiber AB loop (K01: S408E, P409A) (SEQ ID N0:4) and the CD loop (K02: ~V441,K442) (SEQ ID N0:6). We generated pseudotyped viral particles containing fiber proteins that incorporates both of these mutations and then directly demonstrated an effect on viral transduction efficiency. In addition, we have identified a SUBSTITUTE SHEET (RULE 26) novel region of the Ad5 fiber, in the CD loop, that is involved in mediating viral transduction.
All mutants that incorporated amino acid changes within this region displayed a reduction in fiber-mediated gene transfer including KO1+2 (SEQ ID N0:8), K02 (SEQ ID N0:6), K02a (SEQ ID N0:10), K01 (SEQ ID N0:4), K02b (SEQ ID N0:12) and K02c (SEQ 7D N0:14) that resulted in a 1000 to 12-fold reduction. The KO1+2 mutation containing a two amino acid substitution in the A:B loop and a two amino acid deletion in the C:D
loop demonstrated the most potent reduction in gene transfer which is greater than either mutation alone. These data suggest that there may be simply a disturbance in overall structure or a cooperative interaction in CAR binding between these two loop regions.
The K02 (~V441,K442) (SEQ ID N0:6) and K02a (~V441) (SEQ ID NO:10) -mutants showed a. significant decrease in .transduction efficiency, .greater than 1.60-fold. A
portion of this reduction is undoubtedly due to the lower levels of the mutant fiber protein on the viral capsid. However, a significant reduction in transduction efficiency has also been observed for a virus that has the identical K02 mutation introduced genetically into the viral genome. This virus has a full complement of the mi"tant fiber protein on the capsid and still shows a dramatic reduction in transduction efficiency in all cell types tested.
We found here that fiber proteins containing amino acid mutations X509-510 (K04) (SEQ ID N0:18) and 0538-539 (K05) (SEQ ID N0:20) had reduced capability for transducing Hela cells compared to virus particles pseudotyped with wildtype fiber protein.
This suggests that in the Ad5 fiber, these residues on the adjacent monomer are involved in CAR-binding. .. .. ..
The second requirement for an adenovirus that transducer in a cell-type specific manner is the introduction of a novel tropism. The most efficient means is by genetic modification of the fiber gene. Krasnykh et al. { Krasnykh, Dmitriev, et al.
1998 } have shown that the HI loop is an appropriate location in the fiber protein to insert peptides with novel receptor specificities. For example, the cRGD ligand (SEQ ID N0:43) {Pasqualini, Koivunen, et al. 1995 } inserted into the HI loop has been shown toe expand the tropism of Ad both in vitro { Dmitriev, Krasnykh, et al. 1998 } and ih vivo { Reynolds, Dmitriev, et al. 1999 } .
One advantage of the transient transfection/infection system described here is that there is no need for a pseudoreceptor system to propagate virions that do not bind CAR.
CAR binding is needed for efficient viral production. The production of high titer vector stocks containing ablated fiber-CAR interactions is difficult without an alternative cell-binding pathway. Virus production in the transient transfection/infection system involves a single round of replication SUBSTITUTE SHEET (RULE 26) that results in a viral capsid pseudotyped with fiber mutants expressed irc trar~s. It should be possible therefore to more easily test combinations of CAR-binding mutations and targeting ligands for their ability to mediate transduction.

SUBSTITUTE SHEET (RULE 26) TABLE 1. Oligonucleotides used in Ad5 fiber gene mutagenesis.

Fiber ~ Oligonucleotide sequence Fiber expression mutations plasmid pDK01 S408E, Seq. ID 21 5'-ACCACACCAGCTCCAGAGGCTAACTGTAGACTAAATGC-3' P409A
' Seq. ID 22 5'-GCATTTAGTCTACAGTTAGCCTCTGGAGCTGGTGTGTT-3' pDK02 ~V441, Seq. ID 23 5'-ACAGTT'TCAGTTTTGGCCGGCAGTTTGGCTCCAATATC-3' K442 Seq. ID 24 5'-GATATTGGAGCCAAACTGCCGGCCAAAACTGAAACTGT-3' pDK02a ,. S_eq. ID , 5'-ACAGTTTCAGTTTTGGCTAAAGGCAGTTTGGCTCCA-3' .
25 , , Seq. ID 26 5'-TGGAGCCAAACTGCCTTTAGCCAA.AACTGAAACTGT-3' pDK02b ~K442 Seq. ID 5'-GTTTCAGTTTTGGCTGTTGGCAGTTTGGCTCCAATA-3' Seq. ID 5'-TATTGGAGCCAAACTGCCAACAGCCAAAACTGAAAC-3' pDK02c V441A,, , Seq. ID 5'-GTTTCAGTTTTGGCTGCTGCAGGCAGTTTGGCTCCA-3' K442A

Seq. ID 5'-TGGAGCCAAACTGCCTGCAGCAGCCAAAACTGAAAC-3' pDK03 R460E

Seq. II? 5'-GCTCATCTTATTATAGAATTCGACGAAAATGGAGTG-3' Seq. ID 5'-CACTCCATTTTCGTCGAATTCTATAATAAGATGAGC-3' pDK04 OG509, Seq. ID 5'-GCTTATCCAA,AATCTCACACTGCCAAAAGTAACATTGTC-3' K510 Seq. ID 5'-GACAATGTTACTTTTGGCAGTGTGAGATTTTGGATAAGC-3' PDKOS OG538, Seq. ID 5'-CTAACCATTACACTAAACCAGGAAACAGGAGACAC-3' T539 Seq. ID 5'-GTGTCTCCTGTTTCCTGGTTTAGTGTAATGGTTAG-3' Seq. ID 5'-ATAAGATTTGACGAAACTGGAGTGCTACTAAAC-3' Seq. ID 5'-GTTTAGTAGCACTCCAGTTTCGTCAAATCTTAT-3' PDK09 ~ ~ V466H

Seq. ID 5'-TTTGACGAAAATGGACACCTACTAAACAATTCC-3' Seq. ID 5'-GGAATTGTTTAGTAGGTGTCCAGTTTCGTCAAA-3' Seq. I17 5'-AACCTATCAGCTTATGCAAAATCTCACGGTAAA-3' Seq. ID 5'-TTTACCGTGAGATTTTGCATAAGCTGATAGGT-3' tnumbering of amino acid residues as in Xia et al 1994.

SUBSTITUTE SHEET (RULE 26) TABLE 2. Transduction efficiency of pseudotyped AdS.(3gal.~F/F+on Hela cells.
Fiber ~ Fiber mutant RepresentativeMean % wt ( expression avg gal SD)**

plasmid Designation Mutations activity (RLU/~g protein) pDV60 Wildtype None 529882.0 100.0 (0.9) (wt) pDV55 F- Null 266.3 0.1 (0.1)*

pDK01. .... K01 S408E, P409A 7618.0, .. 1.4 (0.8)*-. .. _._ _ .. ..

pDKOl+2 472.3 0.1 (0.0)*
K01+2 S408E, P409A, OV441, pDK02 K02 OV441, K442 3323.7 0.6 (0.2)*

pDK02a K02a OV441 4002.0 0.6 (0.2)*

pDK02b K02b OK442 44399.0 8.3 (0.6)*

pDK02c K02c V441A, K442A 53336.7 8.5 (2.2)*

pDK03 K03 R460E 359229.7 63.3 (9.1)*

pDK04 K04 OG509, K510 212987.7 38.2 (2.8)*

pDK05 K05 OG538, T539 331349.7 58.3 (7.1)*

pDK04+5 499740.3 91.1 (12.1) K04+5 ~G509, K510, OG538, pDK08 K08 N464T 469705.7 92.6 (16.9) pDK09 K09 V466H 391442.7 80.9 (15.1) pDK010 K010 P505A 447260.3 79.6 (6.2) pDK011 K011 0404-581 4521.0 0.8 (0.1)*

RLU, relative light units.
Value represents the average of three wells.

tnumbering of fiber amino acid residues as in Xia et al.
1994.

**% wildtype represents the mean (SD) of the (3gal activity of AdS.(3gal.OF

pseudotyped with each corresponding fiber mutant in 5-6 separate transductions. All values were normalized to wildtype100%.
(pDV60) at *Significantly different from wt fiber using an unpaired, two-tailed t-test analysis, p<0.001 SUBSTITUTE SHEET (RULE 26) Example 2 Description of an AvlnBg Virus Containing a Fiber With the cRGD
Targeting Moiety and the OV441-K442 (K02) CAR-binding Mutation Plasmid Description: The following three plasmids were used to rescue infectious adenoviruses containing modified fibers. pSFloxPRGD is a shuttle plasmid used to incorporate modifed fibers into the Ad genome (Figure 3). It consists of the final 6kb of DNA from the right end of an Av1 genome. A lox site was inserted upstream of the fiber gene. In addition, the Ad5 packaging signal has been inserted near the right inverted terminal repeat (RITR). pAvlhlpr is a helper plasmid that consists of an entire Avl genome with the exception of the right ITR_ (Figure 4).. . It has a reporter gene inserted in the.. E1_ region that .
encodes a nuclear-localized 13-gal protein, the HSV thymidine kinase gene inserted in the E3 region, and a lox site inserted in a location identical to that in p5FloxPRGD.
pCre is a plasmid that constitutively expresses the Cre recombinase. It consists of the Cre gene cloned into the expression plasmid pcDNAl.lzeo+ (Invitrogen). None of these plasmids are capable of producing infectious Ad on their own. But when these three plasmids are cotransfected into a complementing cell line, the Cre protein mediates recombination between the lox sites in p5FloxPRGD and pAvlhlpr, reconstituting a full length Avl genome, which is then capable of producing infectious virus.
Generation of K02 fiber mutations: All amino acid changes were incorporated into the fiber gene using the p5FloxPRGD adenoviral shuttle plasmid as the template. q'his shuttle plasmid encodes a fiber that contains a cRGD peptide sequence, HCDCRGDCFC (SEQ
ID
N0:43), inserted in the HI loop. The cRGD peptide has been shown to bind to a~
integrins on the cell surface. Amino acid residues V441 and K442 in the CD loop of the fiber gene were deleted using the QuickChange Site-Directed Mutagenesis system (Stratagene, La Jolla CA).
Deletion of these residues has been shown in the transient transfectionlinfection system to dramatically inhibit transduction of the Hela cell line which expresses the adenoviral receptor, CAR The resulting shuttle plasmid was called pSK02 (Figure 5).
Generation of adenoviral vectors with CD loop mutations: The mutagenized fiber gene was incorporated into the adenoviral DNA backbone by cre-lox recombination. To do this, the pSK02 shuttle plasmid and the pAvlhlpr helper plasmid were cotransfected with pCre into 633 cells, a cell line that expressed wildtype fiber { Von Seggern, Huang, et al.
2000}. Expression of the Cre recombinase from pCre mediates recombination between lox SUBSTITUTE SHEET (RULE 26) sites in pSK02 and pAvlhlpr, resulting in full length Avl viral DNA, with nuclear (3-Gal transgene in the E1 region. In the 633 complementing cell line, this viral DNA
is capable of being packaged into infectious viral particles containing a mixture of wildtype fiber and mutant fibers. Upon observation of cytopathic effect, the virus was purified by standard CsCl centrifugation procedures. This virus was designated AvlnBgHIRGDK02(633). In order to obtain viral particles containing only the adenoviral-encoded mutant fiber gene with the OV441, K442 mutations (SEQ ID N0:6), this viral preparation was used to infect AE1-2a cells, which do not express fiber {Gorziglia, Kadan, et al. 1996}. Viral particles were purified as above. This virus was designated AvlnBgHIRGDK02.
Comparable fiber incorporation in AvlnB~HIRGDK02: To ensure that the levels of the~mutant fiber on the AvlnBgHIRGDK02 viral particles-were-normal relative to viruses-..
with wildtype fiber levels, Western blot analysis was performed. Equivalent amounts of AvlnBg and AvlnBgHIRGDK02 were subjected to SDS-PAGE. This gel was transferred to a membrane and incubated with rabbit anti-Ad5 fiber and rabbit anti-Ad5 penton polyclonal antisera. The fiber penton ratio on AvlnBgHIRGDK02 viral particles is indistinguishable from that of AvlnBg, demonstrating that there was no effect of the fiber mutations on the level of fiber protein assembled on the viral capsid.
Transduction efficienc~r of AvlnBgHIRGDK02: As shown previously in Example 1, adenoviruses pseudotyped with fiber proteins containing deletions of V441 and K442 in the CD loop are severely affected in their ability mediate gene transfer in the CAR-expressing cellline Hela. In order to test tlie'idea that gene transfer by these mutant viruses can be mediated by alternative ligand/receptor interactions, we tested the ability of the AvlnBgHIRGDK02 virus to transduce HDF, Hela, PC3 and CHO-Kl cell lines (Fig 6). All of these cells lines express a" integrins on the cell surface and, with the exception of Hela, show poor transduction by adenovirus due to a known or presumed deficiency in CAR levels.
Transduction by AvlnBg, which has wildtype fiber, is extremely inefficient in HDF, PC3 .and CHO-KI, as expected (SEQ ID N0:6).,AvlnBgHIRGD, which contains RGD in the HI loop, transduces HDF, PC3 and CHO-Kl with much higher efficiency. However, when cRGD is placed in the context of the V441, K442 deletion, as in the AvlnBgHIRGDK02 virus, transduction efficiency remains inefficient. The levels of AvlnBgHIRGDK02 transduction in HDF and CHO-K1 cells were similar to AvlnBg and lower in PC3 cells.
This reduction in the inability of AvlnBgHIRGDK02 to transduce cells through oc~
integrins is not due to a defect in the RGD targeting moiety. When AvlnBgHIRGDK02 is SUBSTITUTE SHEET (RULE 26) purified from 633 cells, the resulting virus, AvlnBgHIRGDK02 (633) contains a mixture of the wildtype fiber expressed from the 633 cells, and the mutant fiber expressed from the adenoviral genome. This virus, which has both types of fiber on the virion surface, is now able to mediate efficient transduction of HDF cells. This indicates that the RGD in the fiber is able to mediate transduction of HDF cells, even in the context of the V441, K442 deletion.
To further demonstrate that the RGD in the fiber containing the V441, K442 deletion is functional, we performed competition experiments (Fig. 7). HDF cells were transduced with AvIGFPHIRGD in the presence of increasing amounts of a competitor virus AvlnBg, AvlnBgHIRGD or AvlnBgHIRGDK02. The percent of GFP positive cells was then measured. All three competitor viruses successfully inhibited the ability of the ..AyI.GFPHIRGD virus to mediate gene-transfer to comparable degrees.. _ . ., _. . , ' We conclude from these experiments that the RGD retargeting ligand sequence in a fiber containing the V441, K442 deletion is functional in its ability to mediate binding of oc~
integrins (by its ability to inhibit transduction of AvIGFPHIRGD, see Fig. 4) and transduction of cells low in CAR (by its ability to transduce HDF cells when the virus is purified from 633 cells).
In summary, we have described a CD loop mutation, ~V441 K442 (SEQ ID N0:6), that dramatically reduces the ability of viruses pseudotyped with this fiber protein to mediate gene transfer. Here we show that a viral genome containing the V441 K442 deletion and an RGD insertion in the fiber gene can be rescued. This virus was designated " ' " AvlnBgHIRGDKO2: ' The mutant fiber protein is incorporated into ~ the wiral° particles at levels indistinguishable from wildtype. The RGD targeting ligand in AvlnBgHIRGDK02 is functional, as shown in two experiments. First, AvlnBgHIRGDK02 was able to compete AvIGFPHIRGD transduction of HDF cells which are low in CAR and high in a~
integrins.
Second, we showed that retargeting of AvlnBgHIRGDK02 through the RGD targeting ligand in HDF cells can be achieved but was dependent on the propagation of the virus in cells expressing wildtype fiber. We conclude that the V441 K442 deletion has dramatically reduced ability to mediate transduction of CAR-expressing cells and that fibers containing these deletions and alternatives targeting ligands are functional.

SUBSTITUTE SHEET (RULE 26) Example 3 Description Of Adenoviral Vectors Containing a Fiber With the S408E,P409A (K01) CAR-binding Mutation With and Without the cRGD Targeting Moiety Two recombinant adenoviral vectors were prepared that contain the K01 fiber mutation and are designated Av3nBgFK01 and AvlnBgKOlRGD. These vectors contain the KO1 fiber mutation alone or in combination with a cRGD targeting moiety.
The construction of these vectors is described below.
Genetic incorporation of the KO1 fiber mutation in combination with the cRGD
targeting-moiet~generate AvlnBgKOIRGD. All amino acid changes were incorporated into the fiber gene using the p5FloxPRGD adenoviral shuttle plasmid as the template as previously -described un- Example 2: This-- shuttle plasmid encodes a fiber that contains a ~ -cRGD peptide sequence, HCDCRGDCFC, inserted in the fiber HI loop. The eRGD
peptide has been shown to bind to av-integrins on the cell surface. Amino acid residues 408 and 409 in the AB loop of the fiber gene were changed using the Quickchange site-directed mutagenesis system (Stratagene, La Jolla CA). Substitution of these resid~.~
~s has been shown using the transient transfection/infection system to dramatically inhibit transduction of HeLa cells which express the adenoviral receptor, CAR. The resulting shuttle plasmid was called pSK01 (Figure 8). The mutagenized fiber gene and the cRGD targeting moiety were incorporated into the adenoviral DNA backbone by cre-lox recombination. To do this, the pSK01 shuttle plasmid (Figure 8) and the pAvlhlpr plasmid (Figure 4) were co-transfected with pCRE (described in Exaxilple ~2) ~ unto 633' cells, a cell line that expresses 'the wildtype fiber {Von Seggern et al. X000}. Expression of the CRE recombinase from ACRE
mediates recombination between the lox sites in pSK01 and in pAvlhlpr, resulting in full length Avl viral DNA with the nuclear (3-gal transgene in the E1 region to generate AvlnBgKOIRGD.
This virus was initially propagated on 633 cells. After growth on these cells, the virus capsid contained both wildtype and mutant fiber proteins. To obtain viral particles containing only the modified fiber with the KO1 mutation and the RGD moiety, the viral preparation was .:,.
used to infect AE1-Za cells {Gorziglia, Kadan, et al., 1996}, which do not express fiber.
Genetic incorporation of the KO1 fiber mutation into the adenoviral enome. The KO1 mutation alone was incorporated genetically into the adenoviral genome to generate Av3nBgFK0l. The K01 mutation was cloned into a plasmid containing the full-length Av3 adenoviral genome {Gorzigliz, Kadan, et al., 1996} to generate pFLAv3nBgFK01 (Figure 9). Transfections were carried out in 633 cells, and in this fiber complementing cell line, the SUBSTITUTE SHEET (RULE 26) resulting viral DNA containing the KOl mutation is capable of being packaged into infectious viral particles containing a mixture of wildtype fiber and mutant fiber proteins.
Upon observation of CPE, the virus was purified by standard CsCI
centrifugation procedures.
In order to obtain viral particles containing only the adenoviral encoded mutated KO1 fiber protein with the S40~E, P409A mutations, this viral preparation was used to infect AE1-2,a cells, which do not express fiber. Viral particles were purified as described above.
In vitro evaluation of adenoviral vectors containing the K01 fiber mutation.
Several recombinant adenoviral vectors were used in these studies to demonstrate the function and specificity of the KOl fiber mutation and the cRGD targeting moiety. These vectors are described in Table 3.
SUBSTITUTE SHEET (RULE 26) Table 3. Description of recombinant adenoviral vectors used Vector Description Av3nBg An El, E2a, E3-deleted adenoviral vector encoding a nuclear localizing (3-galactosidase Av3nBgFK01 The same as Av3nBg but containing the KO1 mutation in the fiber gene AvI5FHIRGD ~ An E1, E3-deleted vector encoding a nuclear localizing (3-galactosidase and containing a cRGD ligand in the HI loop of fiber AvlnBgKOIRGD ~ An~ El, E3-deleted- vector encoding a ..nuclear .localizing .(3-...
galactosidase and containing both the KO1 fiber mutation and a cRGD ligand in the HI loop.
Transduction efficiency of adenoviral vectors containing AB loop mutations. As shown previously in example 1, adenoviruses pseudotyped with fiber proteins containing the S408E, P409A substitutions in the AB loop are severely affected in their ability to mediate gene transfer in HeLa cells, a CAR-expressing cell line. In order to demonstrate the function and specificity of the K01 mutation and the ability to restore efficient gene transfer by using alternative ligand/receptor interactions, we tested the' ability of the KO1-containing recombinant vectors to transduce various cell types. The four vectors listed in Table 3 were compared for transduction efficiency on HeLa cells, human diploid fibroblasts (HDFs), two different human hepatocellular carcinoma cell lines, Hep3Bs and HepG2s, and a mouse hepatocyte cell line, FL83b. The cells were seeded into the wells of a 24-well dish at 1-2 x 105 cells per well. The next day, the exact number of cells per well was determined for each cell line. by counting a representative well for each cell type. The cells were transduced with various numbers of particles per cell (PPC), in triplicate, using each of the four vectors.
Twenty-four hours after transduction, the cells were stained with X-gal and the percentage of ~i-galactosidase expressing cells was determined by counting cells under the microscope.
Blue cells were counted in six different fields for each well. The total number of cells per field was determined by counting all cells in three fields from only one well, assuming that the total number of cells per field was the same for a given cell type across all wells. The SUBSTITUTE SHEET (RULE 26) results showed that transduction of HeLa, Hep3B, HepG2, and FL83b cells by Av3nBgFK01 was dramatically reduced compared to Av3nBg (Figures 10 and 11). HDFs, which express little or no CAR on their surface, were poorly transduced by both Av3nBg and Av3nBgFK01 (Figure 10B). As expected, efficient transduction of HDFs was observed using the two vectors containing a cRGD ligand in the HI loop, AvI5FHIRGD and AvlnBgKOIRGD
(Figure 10B).
To determine whether the transduction observed in the above study was mediated by fiber-CAR interaction, competition binding experiments using HeLa cells were carried out using either full-length fiber protein or fiber knob as protein competitors (Figure 12).
Competition was carried out by adding 40 ~,g/ml of the full-length fiber or 16 ~.g/ml of the fiber knob directly to . the. cells for 5 minutes at room temperature .prior ..to. infecting...with . .. .
vector. The results showed that either full-length fiber or fiber knob could efficiently block transduction of cells by Av3nBg. However, the low level of transduction of cells by Av3nBgFK01 was not blocked by full-length fiber or fiber knob. This result shows that the low level ~f transduction by Av3nBgFK01 was not mediated by fiber-CAR
interaction.
Similar results were also obtained on human hepatocytes. Fiber competition of Av3nBgFK01 on mouse hepatocytes was not evaluated since transduction in the absence of competition was barely detectable.
In vivo analysis of adenoviral vectors containing the K01 fiber mutation.
Efficient in vivo targeting of adenoviral vectors requires both ablation of the normal tropism and the addition of a new tropism. We~ generated adenoviral vectors which were designed to achieve both of those requirements. (See Table 3.) To ablate the normal tropism, the adenovirus fiber protein was genetically modified to knockout its interaction with the coxsackie-adenovirus receptor (CAR). To retarget the vector to a different receptor, a cRGD
targeting moiety was inserted into the HI Ioop of the fiber knob. In vitro studies demonstrate that adenoviral vectors containing mutations in the fiber knob which knockout its ability to interact with CAR are unable to transduce target cells. One particular fiber knockout mutant, called KO1, reduces transduction of HeLa cells to 1.4 % of the transduction level observed using a vector containing a wild-type fiber. Furthermore, in vitro data has been generated showing that cRGD ligand insertion into the HI loop of the fiber KO1 mutant restores transduction to levels equivalent to that seen with "wild-type' vector. These vectors were next analyzed in vivo using C57BL/6 male mice to assess the extent of liver detargeting and to determine the SUBSTITUTE SHEET (RULE 26) tissue distribution of such vectors following tail vein administration and to determine whether insertion of the cRGD targeting ligand into the HI loop restores liver transduction.
In vivo study design. The study included 5 cohorts of 5 mice each. Adenoviral vectors encoding nuclear targeted (3-galactosidase (nBg) were administered by tail vein injection. The dose was 1 x 1013 particles per kilogram. Mice were sacrificed 3 days after vector administration. Tissues, including liver, lung, heart, kidney, and spleen, were collected. Several assays were utilized to assess the efficiency of liver.transduction and the vector biodistribution and included hexon PCR analysis, (3-gal immunohistochemistry, and the (3-gal Tropix assay. One group of mice received Hanks Balanced Salt Solution (HBSS) instead of adenoviral vector and served as a negative control. A second cohort received Av3nBg, which contains a "wild-type" fiber pi°oteiri and served~as a positive -control. A third ' group received Av3nBgFK0l, a fourth group received AvlnBgKOIHIcRGD, and a fifth group received AvlnBgHIcRGD.
Vector Preparation. Each vector was diluted into sterile HBSS, at a final concentration of 1 x 1012 particles/ml. Mice in-groups 2 through 6 were injected with a volume of 10 ml/kg to achieve a vector dose of 1 x 1013 particles per kg. The HBSS control group received an equivalent dose volume.
Technical Methods. Administration of control and test articles was by bolus tail vein injection. Dose was determined from body weights obtained the day of administration.
Ariirnal's were sacrificed approximately ~°v2 hours after vector.
administration by carbon dioxide asphyxiation. Liver, heart, lung, spleen, and kidney were collected from each animal.
Slices of each tissue approximately 2-3 mm thick were placed in neutral buffered formalin to preserve the sample for ~i-galactosidase immunohistochemistry. For optimal histology, one piece of each liver lobe, a lobe of the lung, a whole kidney, an end cross sectional piece of the spleen, and half of the heart cut longitudinally, were placed into the same container of neutral buffered formalin. The remaining tissue from each organ was placed into a 1 ml cryovial and frozen in dry ice to preserve it for hexon PCR analysis to determine vector content. For the liver, pieces of each lobe were frozen in dry ice to preserve it for hexon PCR analysis and other pieces of each lobe were placed in a "Tropix" vial, and frozen on dry ice.
The results of the immunohistochemical staining for ~i-galactosidase expression showed that Av3nBg, Av3nBgFK0l, AvI5FHIRGD, and AvlnBgKOIRGD all yielded efficient transduction of hepatocytes. Av3nBgFK01 yielded a higher percentage of (3-SUBSTITUTE SHEET (RULE 26) galactosidase expressing cells and a more intense staining than Av3nBg. This result was completely unexpected since Av3nBgFK01 transduction of various cells in culture was dramatically reduced. Evaluation of (3-galactosidase expression in mouse livers by a chemiluminescent assay (Figure 13) confirmed the results of the immunohistochemical staining. Mice that received Av3nBgFK01 demonstrated higher levels of expression than those that received Av3nBg. A measurement of the vector content in hepatocytes was determined by a semi-quantitative hexon PCR assay (Figure 14). The results were consistent with both the immunohistochemical staining and the chemiluminescent assay. The vector content in hepatocytes was approximately 35% higher in the mice that received Av3nBgFK01 than in those that received Av3nBg.
.. _... . Summary. . . . The__ fiber. AB loop mutation" contained . in, Av3nBgFK01, ablates interaction with human and mouse CAR in vitro. However, in vivo this fiber AB
loop mutation behaves quite unexpectantly as it was found to dramatically enhance adenoviral-mediated gene transfer to liver and other organs and results in increasing vector potency.
This fiber modification will be useful for in vivo gene therapy strategies and will allow for lower doses of adenoviral vectors to be used systemically.
Example 4 Increased Liver Transduction Using Av3nBgFK01 In Three Different Mouse Strains _ _ _ The following experiment was done to . ,determine whether the increased liver i transduction observed 'with Av3nBgFKOI compared to- that' using Av3nBg -could be reproduced in various mouse strains.
In vivo analysis of adenoviral vectors containing the KOl fiber mutation.
C57BL/6, Baltic, and CD1 male mice were purchased from Harlan Sprague Dawley (Indianapolis, IN).
When the mice were 5 weeks of age they received either HBSS (vehicle control), Av3nBg, or Av3nBgFK01 via tail vein injection at a dose of 1 x 1013 particles per kg which is approximately 2x1011 particles per mouse. Cohorts of five mice received each treatment.
The vector was diluted to 1 x 1012 particles per ml using Hanks Balanced Salt Solution (HBSS) immediately prior to injection. Three days after vector delivery, the animals were sacrificed and tissues including liver, lung, heart, kidney, and spleen were collected.
Technical methods for Galacto-Light PlusT"" chemiluminescent assay. These tissue samples were used to analyze (3-galactosidase expression using the Galacto-Light PlusT""
chemiluminescent assay (Tropix, Inc., Foster City, CA) systems. Tissue samples were SUBSTITUTE SHEET (RULE 26) collected in lysis matrix tubes containing two ceramic spheres (Bio101, Carlsbad, CA) and frozen on dry ice. The tissues were thawed and 500 p1 of lysis buffer from the Galacto-Light Plus kit was added to each tube. The tissue was homogenized for 30 seconds using a FastPrep System (Bio101, Carlsbad, CA). Liver samples were homogenized for an additional 30 seconds. The [3-galactosidase activity in tissue lysates was assayed according to the manufacturer's instructions.
Results. The results (Figure 15) showed that, on average, Av3nBgFK01 yielded higher levels of liver transduction than Av3nBg in all three mouse strains.
Technical methods for ~i-~alactosidase immunohistochemistry. In addition, slices of each tissue approximately 2-3 mm thick were placed in 10% neutral buffered formalin. After - < fixatioin, these samples-...-were.. ..embedded...in . paraffin, sectioned, and. analyzed.. by.. .....
immunohistochemistry for (3-galactosidase expression. A 1:1200 dilution of a rabbit anti-~3-galactosidase antibody (ICN Pharmaceuticals, Inc.; Costa Mesa, CA) was used in conjunction with a Vectastain ABC kit from Vector Laboratories, Inc. (Burlingame, CA) to visualize positive cells.
Results: The results showed that Av3nBgFK01 yielded higher levels of hepatocyte transduction than Av3nBg in all three mouse strains.
Example 5 Av3nBgFK01 Transduced Primary Mouse Hepatocytes In Culture Relatively Poorly Compared To Av3nBg The transduction efficiencies of Av3nBg and Av3nBgFK01 were evaluated on primary mouse hepatocytes.
Isolation and culturing of primary murine hepatoc es. Primary murine hepatocytes were isolated from adult, male CD-1 mice following a two-step collagenase perfusion via the portal vein, modified from published procedures (Seglen, Methods Cell Biol 1973, 13:29-83; ~"
Liddle et al., J Gastro Hepatol 1998, 13:855-858; Marc et al., Eur J Biochem 2000, 267:963-970). The liver was perfused in situ with Liver Perfusion Medium (Life Technologies, Gaithersburg, MD) followed by treatment with Liver Digest Medium (collagenase-disease;
Life Technologies). The liver was minced, and cells were washed and centrifuged three times in Hepatocyte Wash medium (enriched William's E; Life Technologies) before being SUBSTITUTE SHEET (RULE 26) resuspended in Hepatocye Attachment Medium (Modified William's E, supplemented with I% pen-strep and 5% FBS; Life Technologies). Viability was assessed by trypan blue exclusion. Cells were plated at approximately 1 x 105 viable cells per well on collagen type I-coated 24-well plates and allowed to attach for 2 hr at 37°C in 5%
C02. After 2 hr, unattached cells and media were removed, cells were washed and cultured in HepatoZYME-SFM (Life Technologies). Immunohistochemical staining for albumin confirmed the identity of these cells as hepatocytes.
Transduction efficiencX of Av3nB~FK01 on primary murine hepatocytes.
Approximately 24 hours after plating, the cells were transduced with the adenoviral vectors Av3nBg and Av3nBgFK01 at various numbers of particles per cell, ranging from 0 to _.1.2,500. The.cells were incubated, with adenoviral vector for 1 hour at 37° ,C in a total volume of 0.2 ml of culture medium. Next, the cell monolayers were washed once with PBS, then 1 ml of the appropriate culture medium was added to each well. The cells were incubated for 24 hours to allow for (3-galactosidase expression. The cell monolayers were then fixed and stained with X-Gal for 24 hours. The percentage of transduction was determined by light microscopy by counting the number of transduced, blue cells per total cells in a high-power field with a Nikon CKl microscope; three fields were counted per well. Each vector dose was carried out in triplicate and the average percentage of transduction per high-power field (n=3 wells) was determined. The mean percent transduction obtained from at least three independent experiments was determined.
Results. The results (Figure 16) showed dramatically reduced transduction of primary mouse hepatocytes using Av3nBgFK01 compared to Av3nBg.
REFERENCES
Dmitriev, L, V. Krasnykh, C.R. Miller, M. Wang, E. Kashentseva, G. Mikheeva, N.
Belousova, and D.T. Curiel. 1998. An adenovirus vector with genetically modified fibers demonstrates expanded tropism via utilization of a coxsackievirus and adenovirus receptor-independent cell entry mechanism. J Virol 72:9706-9713.
Gorziglia, M.L, M.J. Kadan, S. Yei, J. Lim, G.M. Lee, R. Luthra, and B.C.
Trapnell. 1996.
Elimination of both E1 and E2 from adenovirus vectors further improves prospects for in vivo human gene therapy. J Virol 70:4173-4178.
Harbury, P.B., T. Zhang, P.S. Kim, and T. Alber. 1993. A switch between two-, three-, and four-stranded coiled coils in GCN4 leucine zipper mutants. Science 262:1401-1407.

SUBSTITUTE SHEET (RULE 26) Horton, R.M., Z.L. Cai, S.N. Ho, and L.R. Pease. 1990. Gene splicing by overlap extension:
tailor-made genes using the polymerase chain reaction. Biotechniques 8:528-535.
Krasnykh, V., I. Dmitriev, G. Mikheeva, C.R. Miller, N. Belousova, and D.T.
Curiel. 1998.
Characterization of an adenovirus vector containing a heterologous peptide epitope in the HI loop of the fiber knob. J Virol 72:1844-1852.
Krasnykh, V.N., G.V. Mikheeva, J.T. Douglas, and D.T. Curiel. 1996. Generation of recombinant adenovirus vectors with modified fibers for altering viral tropism.
JVirol 70:6839-6846.
Michael, S.L, J.S. Hong, D.T. Curiel, and J.A. Engler. 1995. Addition of a short peptide ligand to the adenovirus fiber protein. Gene Ther 2:660-668.
Mittereder,, N.,. K.L.. March, and B.C. Trapnell. 1996. Evaluation.eof ,he, concentration, and.. , bioactivity of adenovirus vectors for gene therapy. J Virol 70:7498-7509.
Pasqualini, R., E. Koivunen, and E. Ruoslahti. 1995. A peptide isolated from phage display libraries is a structural and functional mimic of an RGD-binding site on integrins. J
Cell Biol 130:1189-1196.
Reynolds, P., I. Dmitriev, and D. Curiel. 1999. Insertion of an RGD motif into the HI loop of adenovirus fiber protein alters the distribution of transgene expression of the systemically administered vector. Gene Ther 6:1336-1339.
Stevenson, S.C., M. Rollence, J. Marshall-Neff, and A. McClelland. 1997.
Selective targeting of human cells by a chimeric adenovirus vector containing a modified fiber protein. J , Virol 71:47824790. ~~ - _ J. _ -Stevenson, S.C., M. Rollence, B. White, L. Weaver, and A. McClelland. 1995.
Human adenovirus serotypes 3 and 5 bind to two different cellular receptors via the fiber head domain. J Virol 69:2850-2857.
Von Seggern, D.J., C.Y. Chiu, S.K. Fleck, P.L. Stewart, and G.R. Nemerow.
1999. A helper-independent adenovirus vector with El, E3, and fiber deleted: structure and infectivity of fiberless particles. J Viro173:1601-1608.
Von Seggern, D.J., S. Huang, S.K. Fleck, S.C. Stevenson, and G.R. Nemerow.
2000.
Adenovirus Vector Pseudotyping in Fiber-Expressing Cell Lines: Improved Transduction of Epstein-Barr Virus-Transformed B Cells. J Virol 74:354-362.
Wickham, T.J., R.R. Granados, H.A. Wood, D.A. Hammer, and M.L. Shuler. 1990.
General analysis of receptor-mediated viral attachment to cell surfaces. Biophys J
58:1501-1516.

SUBSTITUTE SHEET (RULE 26) Wickham, T.J., D.M. Segal, P.W. Roelvink, M.E. Carrion, A. Lizonova, G.M. Lee, and I.
Kovesdi. 1996. Targeted adenovirus gene transfer to endothelial and smooth muscle cells by using bispecific antibodies. J Viro170:6831-6838.
Wickham, T.J., E. Tzeng, L.L. Shears, P.W. Roelvink, Y. Li, G.M. Lee, D.E.
Brough, A.
Lizonova, and I. Kovesdi. 1997. Increased in vitro and in vivo gene transfer by adenovirus vectors containing chimeric fiber proteins. J Virol 71:8221-8229.
The disclosures of all patents, patent applications, publications (including published patent applications), depository accession numbers, and database accession numbers referred to in this specification are specifically incorporated herein by reference in their entirety to the same extent as if each such individual , patent, " patent application, publication, depository accession number, and database accession number were specifically and individually indicated to be incorporated in its entirety.

SUBSTITUTE SHEET (RULE 26) SEQUENCE LISTING
<110> Novartis AG
<120> Adenovirus particles with mutagenized fiber proteins <130> 4-31452A
<140> US 09/870,203 <141> 2001-05-30 <160> 43 <170> PatentIn version 3.1 <210> 1 <211> 1746 <212> DNA
<213> Human adenovirus type 5 <220>
<221> CDS
<222>°..~.L1).....- (1746) <223>
<400>

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aaa atgggcaac ggcctc tctctggac gaggcc ggcaacctt acctcc 240 ' Lys MetGlyAsn GlyLeu SerLeuAsp GluAla GlyAsnLeu ThrSer caa aatgtaacc actgtg agc'ccacct ctcaaa aaaaccaag tcaaac 288 Gln AsnValThr ThrVal SerProPro LeuLys LysThrLys SerAsn ata aacctggaa atatct gcacccctc acagtt acctcagaa gcccta 336 Ile AsnLeuGlu IleSer AlaProLeu ThrVal ThrSerGlu AlaLeu act gtggetgcc gccgca cctctaatg gtcgcg ggcaacaca ctcacc 384 Thr ValAlaAla AlaAla ProLeuMet ValAla GlyAsnThr LeuThr atg caatcacag gccccg ctaaccgtg cacgac tccaaactt agcatt 432 Met GlnSerGln AlaPro LeuThrVal HisAsp SerLysLeu SerIle gcc acc caa gga ccc ctc aca gtg tca gaa gga aag cta gcc ctg caa 480 Ala Thr Gln Gly Pro Leu Thr Val Ser Glu Gly Lys Leu Ala Leu Gln SUBSTITUTE SHEET (RULE 26) acatcaggCCCC CtCaCC aCCaCCgat agcagt acccttact atcact 528 ThrSerGlyPro LeuThr ThrThrAsp SerSer ThrLeuThr IleThr gcctCaCCCCCt ctaact actgccact ggtagc ttgggcatt gacttg 576 AlaSerProPro LeuThr ThrAlaThr GlySer LeuGlyIle AspLeu aaagagcccatt tataca caaaatgga aaacta ggactaaag tacggg 624 LysGluProIle TyrThr GlnAsnGly LysLeu GlyLeuLys TyrGly getcctttgcat gtaaca gacgaccta aacact ttgaccgta gcaact 672 AlaProLeuHis ValThr AspAspLeu AsnThr LeuThrVal AlaThr ggtccaggtgtg actatt aataatact tccttg caaactaaa gttact 720 GlyProGlyVal ThrIle AsnAsnThr SerLeu GlnThrLys ValThr ggagccttgggt tttgat tcacaaggc aatatg caacttaat gtagca 768 GlyAlaLeu.Gly ~Phe..Asp..SerGin.GlyAsnMet GlnLeuAsn_ValAla, . _ .
.
, ggaggactaagg attgat tctcaaaac agacgc cttatactt gatgtt 816 GlyGlyLeuArg IleAsp SerGlnAsn ArgArg LeuIleLeu AspVal agttatccgttt gatget caaaaccaa ctaaat ctaagacta ggacag 864 SerTyrProPhe AspAla GlnAsnGln LeuAsn LeuArgLeu GlyGln ggccctcttttt ataaac tcagcccac aacttg gatattaac taeaac 912 GlyProLeuPhe IleAsn SerAlaHis AsnLeu AspIleAsn TyrAsn aaaggcctttac ttgttt acagettca aacaat tccaaaaag cttgag 960 LysGlyLeuTyr LeuPhe ThrAlaSer AsnAsn SerLysLys LeuGlu gttaacctaagc actgcc aaggggttg atgttt gacgetaca gccata 1008 Val.AsnL,euSer ThrAla LysGlyLeu MetPhe AspAlaThr AlaIle z ~

325 ~ ~ 330 335 gccattaatgca ggagat gggcttgaa tttggt tcacctaat gcacca 1056 AlaIleAsnAla GlyAsp GlyLeuGlu PheGly SerProAsn AlaPro aacacaaatccc ctcaaa acaaaaatt ggccat ggcctagaa tttgat 1104 AsnThrAsnPro LeuLys ThrLysIle GlyHis GlyLeuGlu PheAsp tcaaacaagget atggtt cctaaacta ggaact ggccttagt tttgac 1152 SerAsnLysAla MetVal ProLysLeu GlyThr GlyLeuSer PheAsp agcacaggtgcc attaca gtaggaaac aaaaat aatgataag ctaact 1200 SerThrGlyAla IleThr ValGlyAsn LysAsn AsnAspLys LeuThr ttgtggaccaca ccaget ccatctcct aactgt agactaaat gcagag 1248 LeuTrpThrThr ProAla ProSerPro AsnCys ArgLeuAsn AlaGlu aaagatgetaaa ctcact ttggtctta acaaaa tgtggcagt caaata 1296 LysAspAlaLys LeuThr LeuValLeu ThrLys CysGlySer GlnIle SUBSTITUTE SHEET (RULE 26) ctt get acagtt tcagttttg getgtt aaaggcagt ttggetcca ata 1344 Leu Ala ThrVal SerValLeu AlaVal LysGlySer LeuAlaPro Ile tct gga acagtt caaagtget catctt attataaga tttgacgaa aat 1392 Ser Gly ThrVal GlnSerAla HisLeu IleIleArg PheAspGlu Asn gga gtg ctacta aacaattcc ttcctg gacccagaa tattggaac ttt 1440 Gly Val LeuLeu AsnAsnSer PheLeu AspProGlu TyrTrpAsn Phe aga aat ggagat cttactgaa ggcaca gcctataca aacgetgtt gga 1488 Arg Asn GlyAsp LeuThrGlu GlyThr AlaTyrThr AsnAlaVal Gly ttt atg cctaac ctatcaget tatcca aaatctcac ggtaaaact gcc 1536 Phe Met ProAsn LeuSerAla TyrPro LysSerHis GlyLysThr Ala - agt aacatt gtcagt.caa gtttac . aacgga gac_aaaact aaa 1584 _ aaa tta . ._ Lys Ser AsnIle ValSerGln ValTyr LeuAsnGly AspLysThr Lys cct gta acacta accattaca ctaaac ggtacacag gaaacagga gac 1632 Pro Val ThrLeu ThrIleThr LeuAsn GlyThrGln GluThrGly Asp aca act ccaagt gcatactct atgtca ttttcatgg gactggtct ggc 1H80 Thr Thr ProSer A~laTyrSer MetSer PheSerTrp AspTrpSer Gly cac aac tacatt aatgaaata tttgcc acatcctct tacactttt tca 1728 His Asn TyrIle AsnGluIle PheAla ThrSerSer TyrThrPhe Ser tac gcc caa gaa taa 1746 att Tyr Ala Gln Glu Ile 580 _ <210>2 __ ~ . ' .

<211>581 <212>PRT

<213>Human adenovirus type <400>2 Met Arg Ala Arg Pro Ser Glu Asp Thr Phe Asn Pro Val Tyr Pro Lys Tyr Asp Thr Glu Thr Gly Pro Pro Thr Val Pro Phe Leu Thr Pro Pro Phe Val Ser Pro Asn Gly Phe Gln Glu Ser Pro Pro Gly Val Leu Ser Leu Arg Leu Ser Glu Pro Leu Val Thr Ser Asn Gly Met Leu Ala Leu Lys Met Gly Asn Gly Leu Ser Leu Asp Glu Ala Gly Asn Leu Thr Ser SUBSTITUTE SHEET (RULE 26) Gln Asn Val Thr Thr Val Ser Pro Pro Leu Lys Lys Thr Lys Ser Asn Ile Asn Leu Glu Ile Ser Ala Pro Leu Thr Val Thr Ser Glu Ala Leu Thr Val Ala Ala Ala Ala Pro Leu Met Val Ala Gly Asn Thr Leu Thr Met Gln Ser Gln Ala Pro Leu Thr Val His Asp Ser Lys Leu Ser Ile Ala Thr Gln Gly Pro Leu Thr Val Ser Glu Gly Lys Leu Ala Leu Gln ,Thr.Ser Gly ProyLeu~~Thr Thr Thr Asp Ser Ser Thr Leu Thr Ile Thr .

Ala Ser Pro Pro Leu Thr Thr Ala Thr Gly Ser Leu Gly Ile Asp Leu Lys Glu Pro Ile Tyr Thr Gln Asn Gly Lys Leu Gly Leu Lys Tyr Gly Ala Pro Leu His Val Thr Asp Asp Leu Asn Thr Leu Thr Val Ala Thr Gly Pro Gly Val Thr Ile Asn Asn Thr Ser Leu Gln Thr Lys Val Thr Gly Ala Leu Gly Phe Asp Ser Gln Gly~Asn Met Gln Leu Asn Val. Ala Gly Gly Leu Arg Ile Asp Ser Gln Asn Arg Arg Leu Ile Leu Asp Val Ser Tyr Pro Phe Asp Ala Gln Asn Gln Leu Asn Leu Arg Leu Gly Gln Gly Pro Leu Phe Ile Asn Ser Ala His Asn Leu Asp Ile Asn Tyr Asn Lys Gly Leu Tyr Leu Phe Thr Ala Ser Asn Asn Ser Lys Lys Leu Glu Val Asn Leu Ser Thr Ala Lys Gly Leu Met Phe Asp Ala Thr Ala Ile Ala Ile Asn Ala Gly Asp Gly Leu Glu Phe Gly Ser Pro Asn Ala Pro SUBSTITUTE SHEET (RULE 26) Asn Thr Asn Pro Leu Lys Thr Lys Ile Gly His Gly Leu Glu Phe Asp Ser Asn Lys Ala Met Val Pro Lys Leu Gly Thr Gly Leu Ser Phe Asp Ser Thr Gly Ala Ile Thr Val Gly Asn Lys Asn Asn Asp Lys Leu Thr Leu Trp Thr Thr Pro Ala Pro Ser Pro Asn Cys Arg Leu Asn Ala Glu Lys Asp Ala Lys Leu Thr Leu Val Leu Thr Lys Cys Gly Ser Gln Ile Leu Ala Thr Val Ser Val Leu Ala Val Lys Gly Ser Leu Ala Pro Ile Ser Gly Thr Val Gln Ser Ala His Leu Ile Ile Arg Phe Asp Glu Asn 'Gly Val Leu Leu Asn Asn Ser Phe Leu Asp Pro Glu Tyr Trp Asn Phe Arg Asn Gly Asp Leu Thr Glu Gly Thr Ala Tyr Thr Asn Ala Val Gly Phe Met Pro Asn Leu Ser Ala Tyr Pro Lys Ser His Gly Lys Thr Ala Lys Ser Asn Tle Val Ser Gln Val Tyr Leu Asn Gly Asp Lys Thr Lys Pro Val Thr Leu Thr Ile Thr Leu Asn Gly Thr Gln Glu Thr Gly Asp Thr Thr Pro Ser Ala Tyr Ser Met Ser Phe Ser Trp Asp Trp Ser Gly His Asn Tyr Ile Asn Glu Ile Phe Ala Thr Ser Ser Tyr Thr Phe Ser Tyr Ile Ala Gln Glu <210> 3 <211> 1746 <212> DNA
<213> Artificial Sequence SUBSTITUTE SHEET (RULE 26) <220>
<223> Codes for a mutated Human Adenovirus type 5 fiber protein.
<220>
<221> CDS
<222> (1)..(1746) <223>
<220>
<221> mutation <222> (1222)..(1227) <223>
<400>

atg aagcgcgca agaccg tctgaagat accttc aaccecgtg tatcca 48 Met LysArgAla ArgPro SerGluAsp ThrPhe AsnProVal TyrPro tat gacacggaa accggt cctccaact gtgcc_ttttcttact cctccc . 96 ~ ~ ~

Tyr AspThrGlu ThrGly ProProThr ValPro PheLeuThr ProPro ttt gtatccccc aatggg tttcaagag agtCCC CCtggggta CtCtct 144 Phe ValSerPro AsnGly PheGlnGlu SerPro ProGlyVal LeuSer ttg cgcctatcc gaacct ctagttacc tccaat ggcatgctt gcgctc 192 Leu ArgLeuSer Glu.Pro LeuValThr SerAsn GlyMetLeu AlaLeu aaa atgggcaac ggcctc tctctggac gaggcc ggcaacctt acctcc 240 Lys MetGlyAsn GlyLeu SerLeuAsp GluAla GlyAsnLeu ThrSer caa aatgtaacc actgtg agcccacct ctcaaa aaaaccaag tcaaac 288 Gln AsnValThr ThrVal SerProPro LeuLys LysThrLys SerAsn ata aacctggaa atatct gcacccctc acagtt acctcagaa gcccta 336 Ile AsnLeuGlu IleSer AlaProLeu ThrVal ThrSerGlu AlaLeu act gtggetgcc gccgca cctctaatg gtcgcg ggcaacaca ctcacc 384 Thr ValAlaAla AlaAla ProLeuMet Va1Ala GlyAsnThr LeuThr atg caatcacag gccccg ctaaccgtg cacgac tccaaactt agcatt 432 Met GlnSerGln AlaPro LeuThrVal HisAsp SerLysLeu SerIle gcc acccaagga cccctc acagtgtca gaagga aagctagcc ctgcaa 480 Ala ThrGlnGly ProLeu ThrValSer GluGly LysLeuAla LeuGln 145 150 ' ~- 155 160 aca tcaggcccc ctcacc accaccgat agcagt acccttact atcact 528 Thr SerGlyPro LeuThr ThrThrAsp SerSer ThrLeuThr IleThr gcc tCaCCCCCt ctaact actgccact ggtagc ttgggcatt gacttg 576 Ala SerProPro LeuThr ThrAlaThr GlySer LeuGlyIle AspLeu aaa gagcccatt tataca caaaatgga aaacta ggactaaag tacggg 624 Lys GluProIle TyrThr GlnAsnGly LysLeu GlyLeuLys TyrGly SUBSTITUTE SHEET (RULE 26) get cctttg catgtaaca gacgaccta aacact ttgaccgta gcaact 672 Ala ProLeu HisValThr AspAspLeu AsnThr LeuThrVal AlaThr ggt ccaggt gtgactatt aataatact tccttg caaactaaa gt~tact 720 Gly ProGly ValThrIle AsnAsnThr SerLeu GlnThrLys ValThr gga gccttg ggttttgat tcacaaggc aatatg caacttaat gtagca 768 Gly AlaLeu GlyPheAsp SerGlnGly AsnMet GlnLeuAsn ValAla gga ggacta aggattgat tctcaaaac agacgc cttatactt gatgtt 816 Gly GlyLeu ArgIleAsp SerGlnAsn ArgArg LeuIleLeu AspVal agt tatccg tttgatget caaaaccaa ctaaat ctaagacta ggacag 864 Ser TyrPro PheAspAla GlnAsnGln LeuAsn LeuArgLeu GlyGln ggc cctctt tttataaac~tcagcccac aacttg gatattaac tacaac 912 ~

Gly ProLeu PheIleAsn SerAlaHis AsnLeu AspIleAsn TyrAsn aaa ggcctt tacttgttt acagettca aacaat tccaaaaag cttgag 960 Lys GlyLeu TyrLeuPhe ThrAlaSer AsnAsn SerLysLys LeuGlu gtt aaccta agcactgcc aaggggttg~atgttt gacgetaca gccata 1008 Val AsnLeu SerThrAla LysGlyLeu MetPhe AspAlaThr AlaIle gcc attaat gcaggagat gggcttgaa tttggt tcacctaat gcacca 1056 Ala IleAsn AlaGlyAsp GlyLeuGlu PheGly SerProAsn AlaPro aac acaaat cccctcaaa acaaaaatt ggccat ggcctagaa tttgat 1104 Asn ThrAsn ProLeuLys ThrLysIle GlyHis GlyLeuGlu PheAsp tca aacaag getatggtt cctaaacta ggaact ggccttagt tttgac 1152 Ser AsnLys AlaMetVal ProLysLeu GlyThr GlyLeuSer PheAsp , agc acaggt gccattaca gtaggaaac aaaaat aatgataag ctaact 1200 Ser ThrGly AlaIleThr ValGlyAsn LysAsn AsnAspLys LeuThr ttg tggacc acaccaget ccagagget aactgt agactaaat gcagag 1248 Leu TrpThr ThrProAla ProGluAla AsnCys ArgLeuAsn AlaGlu aaa gatget aaactcact ttggtctta acaaaa tgtggcagt caaata 1296 Lys AspAla LysLeuThr LeuValLeu ThrLys CysGly~SerGlnIle ctt getaca gtttcagtt ttggetgtt aaaggc agtttgget ccaata 1344 Leu AlaThr ValSerVal LeuAlaVal LysGly SerLeuAla ProIle tct ggaaca gttcaaagt getcatctt attata agatttgac gaaaat 1392 Ser GlyThr ValGlnSer AlaHisLeu IleIle ArgPheAsp GluAsn gga gtg cta cta aac aat tcc ttc ctg gac cca gaa tat tgg aac ttt 1440 SUBSTITUTE SHEET (RULE 26) Gly Val Leu Leu Asn Asn Ser Phe Leu Asp Pro Glu Tyr Trp Asn Phe agaaat ggagat cttactgaa ggcaca gcctataca aacgetgtt gga 1488 ArgAsn GlyAsp LeuThrGlu GlyThr AlaTyrThr AsnAlaVal Gly tttatg cctaac ctatcaget tatcca aaatctcac ggtaaaact gcc 1536 PheMet ProAsn LeuSerAla TyrPro LysSerHis GlyLysThr Ala aaaagt aacatt gtcagtcaa gtttac ttaaacgga gacaaaact aaa 1584 LysSer AsnIle ValSerGln ValTyr LeuAsnGly AspLysThr Lys cctgta acacta accattaca ctaaac ggtacacag gaaacagga gac 1632 ProVal ThrLeu ThrIleThr LeuAsn GlyThrGln GluThrGly Asp acaact ccaagt gcatactct atgtca ttttcatgg gactggtct ggc 1680 ThrThr ProSer AlaTyrSer MetSer PheSerTrp AspTrpSer Gly 545. 550 . 555 _ 560 _ _, cacaac tacatt aatgaaata tttgcc acatcctct tacactttt tca 1728 HisAsn TyrIle AsnGluIle PheAla ThrSerSer TyrThrPhe Ser tacatt gcccaa gaataa 1746 TyrIle AlaGln Glu <210> 4 <221> 581 <222> PRT
<213> Artificial Sequence <220>
<223> Codes for a mutated Human Adenovirus type 5 fiber protein.
<400> 4 Met Lys Arg Ala Arg Pro Ser Glu Asp Thr Phe Asn Pro Val Tyr Pro Tyr Asp Thr Glu Thr Gly Pro Pro Thr Val Pro Phe Leu Thr Pro Pro Phe Val Ser Pro Asn Gly Phe Gln Glu Ser Pro Pro Gly Val Leu Ser Leu Arg Leu Ser Glu Pro Leu Val Thr Ser Asn Gly Met Leu Ala Leu 50 - ' 55 60 Lys Met Gly Asn Gly Leu Ser Leu Asp Glu Ala Gly Asn Leu Thr Ser Gln Asn Val Thr Thr Val Ser Pro Pro Leu Lys Lys Thr Lys Ser Asn Ile Asn Leu Glu Ile Ser Ala Pro Leu Thr Val Thr Ser Glu Ala Leu SUBSTITUTE SHEET (RULE 26) Thr Val Ala Ala Ala Ala Pro Leu Met Val Ala Gly Asn Thr Leu Thr Met Gln Ser Gln Ala Pro Leu Thr Val His Asp Ser Lys Leu Ser Ile Ala Thr Gln Gly Pro Leu Thr Val Ser Glu Gly Lys Leu Ala Leu Gln Thr Ser Gly Pro Leu Thr Thr Thr Asp Ser Ser Thr Leu Thr Ile Thr Ala Ser Pro Pro Leu Thr Thr Ala Thr Gly Ser Leu Gly Ile Asp Leu Lys Glu Pro Ile Tyr Thr Gln Asn Gly Lys Leu Gly Leu Lys Tyr Gly Ala Pro Leu His Val Thr Asp Asp Leu Asn Thr Leu Thr Val Ala Thr 210 23.5 220 Gly Pro Gly Val Thr Ile Asn Asn Thr Ser Leu Gln Thr Lys Val Thr Gly Ala Leu Gly Phe Asp Ser Gln Gly Asn Met Gln Leu Asn Val Ala Gly Gly Leu Arg Ile Asp Ser Gln Asn Arg Arg Leu Ile Leu Asp Val Ser Tyr Pro Phe Asp Ala Gln Asn Gln Leu Asn Leu Arg Leu Gly Gln Gly Pro Leu Phe Ile Asn Ser Ala His Asn Leu Asp Ile Asn Tyr Asn Lys Gly Leu Tyr Leu Phe Thr Ala Ser Asn Asn Ser Lys Lys Leu Glu Val Asn Leu Ser Thr Ala Lys Gly Leu Met Phe Asp Ala Thr Ala Ile Ala Ile Asn Ala Gly Asp Gly Leu Glu Phe Gly Ser Pro Asn Ala Pro Asn Thr Asn Pro Leu Lys Thr Lys Ile Gly His Gly Leu Glu Phe Asp SUBSTITUTE SHEET (RULE 26) Ser Asn Lys Ala Met Val Pro Lys Leu Gly Thr Gly Leu Ser Phe Asp Ser Thr Gly Ala Ile Thr Val Gly Asn Lys Asn Asn Asp Lys Leu Thr Leu Trp Thr Thr Pro Ala Pro Glu Ala Asn Cys Arg Leu Asn Ala Glu Lys Asp Ala Lys Leu Thr Leu Val Leu Thr Lys Cys Gly Ser Gln Tle Leu Ala Thr Val Ser Val Leu Ala Val Lys Gly Ser Leu Ala Pro Ile Ser Gly Thr Val Gln Ser Ala His Leu Ile Ile Arg Phe Asp Glu Asn Gly Val Leu Leu Asn Asn Ser Phe Leu Asp Pro Glu Tyr Trp Asn Phe Arg Asn Gly Asp Leu Thr Glu Gly Thr Ala Tyr Thr Asn Ala Val Gly Phe Met Pro Asn Leu Ser Ala Tyr Pro Lys Ser His Gly Lys Thr Ala Lys Ser Asn Ile Val Ser Gln Val Tyr Leu Asn Gly Asp Lys Thr Lys Pro Val Thr Leu Thr Ile Thr Leu Asn Gly Thr Gln Glu Thr Gly Asp Thr Thr Pro Ser Ala Tyr Ser Met Ser Phe Ser Trp Asp Trp Ser Gly His Asn Tyr Ile Asn Glu Ile Phe Ala Thr Ser Ser Tyr Thr Phe Ser Tyr Ile Ala Gln Glu <210> 5 <211> 1740 <212> DNA
<213> Artificial Sequence <220>
<223> Codes for a mutated Human Adenovirus type 5 fiber protein. Nucle otides corresponding to amino acids 441(V) & 442(x) of wild-type fiber were deleted.
<220>
SUBSTITUTE SHEET (RULE 26) <221> CDS
<222> (1)..(1740) <223>
<400>

atg aagcgcgca agaccg tctgaagat accttcaac cccgtg tatcca 48 Met LysArgAla ArgPro SerGluAsp ThrPheAsn ProVal TyrPro tat gacacggaa accggt cctccaact gtgcctttt cttact cctccc 96 Tyr AspThrGlu ThrGly ProProThr ValProPhe LeuThr ProPro ttt gtatccccc aatggg tttcaagag agtccccct ggggta ctctct 144 Phe ValSerPro AsnGly PheGlnGlu SerProPro GlyVal LeuSer ttg cgcctatcc gaacct ctagttacc tccaatggc atgctt gcgctc 192 Leu ArgLeuSer GluPro LeuValThr SerAsnGly MetLeu AlaLeu aaa atgggcaac~ggcctc tctctggac gaggccggc aacctt acctcc ~

Lys MetGlyAsn GlyLeu SerLeuAsp GluAlaGly AsnLeu ThrSer caa aatgtaacc actgtg agcccacct ctcaaaaaa accaag tcaaac 288 Gln AsnValThr ThrVal SerProPro LeuLysLys ThrLys SerAsn ata aacctggaawatatct gcacccctc acagttacc tcagaa gcccta 336 Ile AsnLeuGlu IleSer AlaProLeu ThrValThr SerGlu AlaLeu act gtggetgcc gccgca cctctaatg gtcgcgggc aacaca ctcacc 384 Thr ValAlaAla AlaAla ProLeuMet ValAlaGly AsnThr LeuThr atg caatcacag gccccg ctaaccgtg cacgactcc aaactt agcatt 432 Met GlnSerGln AlaPro LeuThrVal HisAspSer LysLeu SerIle gcc acccaagga cccctc acagtgtca gaaggaaag ctagcc ctgcaa 480 Ala ThrGlnGly ProLeu ThrValSer GluGlyLys LeuAla LeuGln aca tcaggCCCC CtCaCC aCCaCCgat agcagtacc cttact atcact 528 Thr SerGlyPro LeuThr ThrThrAsp SerSerThr LeuThr IleThr gcc tcaccccct ctaact actgccact ggtagcttg ggcatt gacttg 576 Ala SerProPro LeuThr ThrAlaThr GlySerLeu GlyIle AspLeu aaa gagcccatt tataca caaaatgga aaactagga ctaaag tacggg 624 Lys GluProIle TyrThr GlnAsnGly LysLeuGly LeuLys TyrGly get cctttgcat gtaaca gacgaccta aacactttg accgta gcaact 672 Ala ProLeuHis ValThr AspAspLeu AsnThrLeu ThrVal AlaThr ggt ccaggtgtg actatt aataatact tccttgcaa actaaa gttact 720 Gly ProGlyVal ThrIle AsnAsnThr SerLeuGln ThrLys ValThr gga gccttgggt tttgat tcacaaggc aatatgcaa cttaat gtagca 768 SUBSTITUTE SHEET (RULE 26) Gly AlaLeuGly PheAsp SerGlnGly AsnMetGln LeuAsn ValAla gga ggactaagg attgat tctcaaaac agacgcctt atactt gatgtt 816 Gly GlyLeuArg IleAsp SerGlnAsn ArgArgLeu IleLeu AspVal agt tatccgttt gatget caaaaccaa ctaaatcta agacta ggacag 864 Ser TyrProPhe AspAla GlnAsnGln LeuAsnLeu ArgLeu GlyGln ggc cctcttttt ataaac tcagcccac aacttggat attaac tacaac 912 Gly ProLeuPhe IleAsn SerAlaHis AsnLeuAsp IleAsn TyrAsn aaa ggcctttac ttgttt acagettca aacaattcc aaaaag cttgag 960 Lys GlyLeuTyr LeuPhe ThrAlaSer AsnAsnSer LysLys LeuGlu gtt aacctaagc actgcc aaggggttg atgtttgac getaca gccata 1008 Val AsnLeuSer ThrAla LysGlyLeu MetPheAsp AlaThr AlaIle gcc attaatgca ggagat gggcttgaa tttggttca cctaat gcacca 1056 Ala IleAsnAla GlyAsp GlyLeuGIu PheGlySer ProAsn AlaPro aac acaaatccc ctcaaa acaaaaatt ggccatggc ctagaa tttgat 1104 Asn ThrAsnPro LeuLys ThrLysIle GlyHisGly LeuGlu PheAsp tca aacaagget atggtt cctaaacta ggaactggc cttagt tttgac 1152 Ser AsnLysAla MetVal ProLysLeu GlyThrGly LeuSer PheAsp agc acaggtgcc attaca gtaggaaac aaaaataat gataag ctaact 1200 Ser ThrGlyAla IleThr ValGlyAsn LysAsnAsn AspLys LeuThr ttg tggaccaca ccaget ccatctcct aactgtaga ctaaat gcagag 1248 Leu TrpThrThr ProAla ProSerPro AsnCysArg LeuAsn AlaGlu aaa gatgetaaa ctcact ttggtctta acaaaatgt ggcagt caaata 1296 Lys AspAlaLys LeuThr LeuValLeu ThrLysCys GlySer GlnTle ctt getacagtt tcagtt ttggccggc agtttgget ccaata tctgga 1344 Leu AlaThrVal SerVal LeuAlaGly SerLeuAla ProIle SerGly aca gttcaaagt getcat cttattata agatttgac gaaaat ggagtg 1392 Thr ValGlnSer AlaHis LeuIleIle ArgPheAsp GluAsn GlyVal ' cta ctaaacaat tcc'ttc ctggaccca gaatattgg aacttt agaaat 1440 Leu LeuAsnAsn SerPhe LeuAspPro GluTyrTrp AsnPhe ArgAsn gga gatcttact gaaggc acagcctat acaaacget gttgga tttatg 1488 Gly AspLeuThr GluGly ThrAlaTyr ThrAsnAla ValGly PheMet.

cct aacctatca gettat ccaaaatct cacggtaaa actgcc aaaagt 1536 Pro AsnLeuSer AlaTyr ProLysSer HisGlyLys ThrAla LysSer SUBSTITUTE SHEET (RULE 26) aac attgtcagt caagtt tacttaaac gga aaa actaaa cctgta 1584 gac Asn IleValSer GlnVal TyrLeuAsn Gly Lys ThrLys ProVal Asp aca ctaaccatt acacta aacggtaca cag aca ggagac acaact 1632 gaa Thr LeuThrIle ThrLeu AsnGlyThr Gln Thr GlyAsp ThrThr Glu cca agtgcatac tctatg tcattttca tgg tgg tctggc cacaac 1680 gac Pro SerAlaTyr SerMet SerPheSer Trp Trp SerGly HisAsn Asp tac attaatgaa atattt gccacatcc tct act ttttca tacatt 1728 tac Tyr IleAsnGlu IlePhe AlaThrSer Ser Thr PheSer TyrIle Tyr gcc caagaataa Ala GlnGlu <210> 6 , <211> 579 <212> PRT
<213> Artificial Sequence <220>
<223> Codes for a mutated Human Adenovirus type 5 fiber protein. Nucle otides corresponding to amino acids 441(V) & 442(x) of wild-type fiber were deleted.
<400> 6 Met Lys Arg Ala Arg Pro Ser Glu Asp Thr Phe Asn Pro Val Tyr Pro Tyr Asp Thr Glu Thr Gly Pro Pro Thr Val Pro Phe Leu Thr Pro Pro Phe Val Ser Pro Asn Gly Phe Gln Glu Ser Pro Pro GlyyVal Leu.Ser Leu Arg Leu Ser Glu Pro Leu Val Thr Ser Asn Gly Met Leu Ala Leu Lys Met Gly Asn Gly Leu Ser Leu Asp Glu Ala Gly Asn Leu Thr Ser Gln Asn Val Thr Thr Val Ser Pro Pro Leu Lys Lys Thr Lys Ser Asn Ile Asn Leu Glu Ile Ser Ala Pro Leu Thr Val Thr Ser Glu Ala Leu Thr Val Ala Ala Ala Ala Pro Leu Met Val Ala Gly Asn Thr Leu Thr Met Gln Ser Gln Ala Pro Leu Thr Val His Asp Ser Lys Leu Ser Ile SUBSTITUTE SHEET (RULE 26) Ala Thr Gln Gly Pro Leu Thr Val Ser Glu Gly Lys Leu Ala Leu Gln Thr Ser Gly Pro Leu Thr Thr Thr Asp Ser Ser Thr Leu Thr Ile Thr A1a Ser Pro Pro Leu Thr Thr Ala Thr Gly Ser Leu Gly Ile Asp Leu Lys Glu Pro Ile Tyr Thr Gln Asn Gly Lys Leu Gly Leu Lys Tyr Gly Ala Pro Leu His Val Thr Asp Asp Leu Asn Thr Leu Thr Val Ala Thr Gly Pro Gly Val Thr Ile Asn Asn Thr Ser Leu Gln Thr Lys Val Thr Gly Ala Leu Gly Phe Asp Ser Gln Gly Asn Met Gln Leu Asn Val Ala Gly Gly Leu Arg.Ile Asp Ser Gln Asn Arg Arg Leu Ile Leu Asp Val Ser Tyr Pro Phe Asp Ala Gln Asn Gln Leu Asn Leu Arg Leu Gly Gln Gly Pro Leu Phe Ile Asn Ser Ala His Asn Leu Asp Ile Asn Tyr Asn Lys Gly Leu Tyr Leu Phe Thr Ala Ser Asn Asn Ser Lys Lys Leu Glu 305 3l0 315 320 Val Asn Leu Ser Thr Ala Lys Gly Leu Met Phe Asp Ala Thr Ala Ile Ala Ile Asn Ala Gly Asp Gly Leu Glu Phe Gly Ser Pro Asn Ala Pro Asn Thr Asn Pro Leu Lys Thr Lys Ile Gly His Gly Leu Glu Phe Asp 355 360 ° 365 Ser Asn Lys Ala Met Val Pro Lys Leu Gly Thr Gly Leu Ser Phe Asp 370 375 380 .
Ser Thr Gly Ala Ile Thr Val Gly Asn Lys Asn Asn Asp Lys Leu Thr Leu Trp Thr Thr Pro Ala Pro Ser Pro Asn Cys Arg Leu Asn Ala Glu SUBSTITUTE SHEET (RULE 26) Lys Asp Ala Lys Leu Thr Leu Val Leu Thr Lys Cys Gly Ser Gln Ile Leu Ala Thr Val Ser Val Leu Ala Gly Ser Leu Ala Pro Ile Ser Gly Thr Val Gln Ser Ala His Leu Ile Ile Arg Phe Asp Glu Asn Gly Val Leu Leu Asn Asn Ser Phe Leu Asp Pro Glu Tyr Trp Asn Phe Arg Asn Gly Asp Leu Thr Glu Gly Thr Ala Tyr Thr Asn Ala Val Gly Phe Met Pro Asn Leu Ser Ala Tyr Pro Lys Ser His Gly Lys Thr Ala Lys Ser Asn Ile Val Ser Gln Val Tyr Leu Asn Gly Asp Lys Thr Lys Pro Val Thr Leu Thr Ile Thr Leu Asn Gly Thr Gln Glu Thr Gly Asp Thr Thr Pro Ser Ala Tyr Ser Met Ser Phe Ser Trp Asp Trp Ser Gly His Asn Tyr Ile Asn Glu Ile Phe Ala Thr Ser Ser Tyr Thr Phe Ser Tyr Ile Ala Gln Glu <210> 7 <211> 1740 <212> DNA
<213> Artificial Sequence <220>
<223> Codes for a mutated Human Adenovirus type 5 fiber protein. Combin anon of the mutant fiber encoded in Seq ID: #3 & 5.
<220>
<221> CDS
<222> (1)..(1740) <223>
<400> 7 atg aag cgc gca aga ccg tct gaa gat acc ttc aac ccc gtg tat cca 48 Met Lys Arg Ala Arg Pro Ser Glu Asp Thr Phe Asn Pro Val Tyr Pro SUBSTITUTE SHEET (RULE 26) tatgacacggaa accggt cctccaact gtgcctttt cttact cctccc, 96 TyrAspThrGlu ThrGly ProProThr ValProPhe LeuThr ProPro tttgtatccccc aatggg tttcaagag agtccccct ggggta ctctct 144 PheValSerPro AsnGly PheGlnGlu SerProPro GlyVal LeuSer ttgcgcctatcc gaacct ctagttacc tccaatggc atgctt gcgctc 192 LeuArgLeuSer GluPro LeuValThr SerAsnGly MetLeu AlaLeu aaaatgggcaac ggcctc tctctggac gaggccggc aacctt acctcc 240 LysMetGlyAsn GlyLeu SerLeuAsp GluAlaGly AsnLeu ThrSer caaaatgtaacc actgtg agcccacct ctcaaaaaa accaag tcaaac 288 GlnAsnValThr ThrVal SerProPro LeuLysLys ThrLys SerAsn ataaacctggaa atatCt gCaCCCCtC aCagttacc tcagaa gcccta 336 IleAsnLeuGlu IleSer AlaProLeu ThrValThr SerG1u AlaLeu.,.

actgtggetgcc gccgca cctctaatg gtcgcgggc aacaca ctcacc 384 ThrValAlaAla AlaAla ProLeuMet ValAlaGly AsnThr LeuThr atgcaatcacag gccccg ctaaccgtg cacgactcc aaactt agcatt 432 MetGlnSerGln AlaPro LeuThrVal His,AspSer LysLeu SerIle gccacccaagga cccctc acagtgtca gaaggaaag ctagcc ctgcaa 480 AlaThrGlnGly ProLeu ThrValSer GluGlyLys LeuAla LeuGln 145 150 155 , 160 acatcaggcccc ctcacc accaccgat agcagtacc cttact atcact 528 ThrSerGlyPro LeuThr ThrThrAsp SerSerThr LeuThr IleThr gcctcaccccct ctaact actgccact ggtagcttg ggcatt gacttg 576 AlaSerProPro LeuThr ThrAlaThr GlySerLeu GlyIle AspLeu " , aaagagcccatt tataca caaaatgga aaactagga ctaaag tacggg 624 LysGluProIle TyrThr GlnAsnGly LysLeuGly LeuLys TyrGly getcctttgcat gtaaca gacgaccta aacactttg accgta gcaact 672 AlaProLeuHis ValThr AspAspLeu AsnThrLeu ThrVal AlaThr ggtccaggtgtg actatt aataatact tccttgcaa actaaa gttact 720 GlyProGlyVal ThrIle AsnAsnThr SerLeuGln ThrLys ValThr ggagccttgggt tttgat tcacaaggc aatatgcaa cttaat gtagca 768 GlyAlaLeuGly PheAsp SerGlnGly AsnMetGln LeuAsn ValAla ggaggactaagg attgat tctcaaaac agacgcctt atactt gatgtt 816 GlyGlyLeuArg IleAsp SerGlnAsn ArgArgLeu IleLeu AspVal agttatccgttt gatget caaaaccaa ctaaatcta agacta ggacag 864 SerTyrProPhe AspAla GlnAsnGln LeuAsnLeu ArgLeu GlyGln SUBSTITUTE SHEET (RULE 26) ggc cctcttttt ataaac tcagcccac aacttg gatattaac tacaac 912 Gly ProLeuPhe IleAsn SerAlaHis AsnLeu AspIleAsn TyrAsn aaa ggcctttac ttgttt acagettca aacaat tccaaaaag cttgag 960 Lys GlyLeuTyr LeuPhe ThrAlaSer AsnAsn SerLysLys LeuGlu gtt aacctaagc actgcc aaggggttg atgttt gacgetaca gccata 1008 Val AsnLeuSer ThrAla LysGlyLeu MetPhe AspAlaThr AlaIle gcc attaatgca ggagat gggcttgaa tttggt tcacctaat gcacca 1056 Ala IleAsnAla GlyAsp GlyLeuGlu PheGly SerProAsn AlaPro aac acaaatccc ctcaaa acaaaaatt ggccat ggcctagaa tttgat 1104 Asn ThrAsnPro LeuLys ThrLysIle GlyHis GlyLeuGlu PheAsp tca aacaagget atggtt cc,taaacta ggaact ggccttagt tttgac 1152 ~

Ser AsnLysAla MetVal ProLysLeu GlyThr GlyLeuSer PheAsp agc acaggtgcc attaca gtaggaaac aaaaat aatgataag ctaact 1200 Ser ThrGlyAla IleThr ValGlyAsn LysAsn AsnAspLys LeuThr ttg tggaccaca ccaget ccagagget aactgt agactaaat gcagag 1248 Leu TrpThrThr ProAla ProGluAla AsnCys ArgLeuAsn AlaGlu ..

aaa gatgetaaa ctcact ttggtctta acaaaa tgtggcagt caaata 1296 Lys AspAlaLys LeuThr LeuValLeu ThrLys CysGlySer GlnIle ctt getacagtt tcagtt ttggccggc agtttg getccaata tctgga 1344 Leu AlaThrVal SerVal LeuAlaGly SerLeu AlaProIle SerGly ,, gttcaaagt getcat ctt.attata agattt gacgaaaat ggagtg 1392 aca Thr ValGlnSer AlaHis LeuIleIle ArgPhe AspGluAsn GlyVa l ctactaaacaat tccttc ctggaccca gaatattgg aactttaga aat 1440 LeuLeuAsnAsn SerPhe LeuAspPro GluTyrTrp AsnPheArg Asn ggagatcttact gaaggc acagcctat acaaacget gttggattt atg 1488 GlyAspLeuThr GluGly ThrAlaTyr ThrAsnAla ValGlyPhe Met cctaacctatca gettat ccaaaatct cacggtaaa actgccaaa agt 1536 ProAsnLeuSer AlaTyr ProLysSer HisGlyLys ThrAlaLys Ser aacattgtcagt caagtt tacttaaac ggagacaaa actaaacct gta 1584 AsnIleValSer GlnVal TyrLeuAsn GlyAspLys ThrLysPro Val 515 520 . 525 acactaaccatt acacta aacggtaca caggaaaca ggagacaca act 1632 ThrLeuThrIle ThrLeu AsnGlyThr GlnGluThr GlyAspThr Thr ccaagtgcatac tctatg tcattttca tgggactgg tctggccac aac 1680 ProSerAlaTyr SerMet SerPheSer TrpAspTrp SerGlyHis Asn SUBSTITUTE SHEET (RULE 26) tac att aat gaa ata ttt gcc aca tcc tct tac act ttt tca tac att 1728 Tyr Ile Asn Glu Ile Phe Ala Thr Ser Ser Tyr Thr Phe Ser Tyr Ile gcc caa gaa taa 1740 Ala Gln Glu <210> 8 <211> 579 <212> PRT
<213> Artificial Sequence <220>
<223> Codes for a mutated Human Adenovirus type 5 fiber protein. Combin ation of the mutant fiber encoded in Seq ID: #3 & 5.
<400> 8 Met Lys Arg Ala Arg.Pro Ser Glu Asp Thr Phe Asn Pro Val Tyr Pro Tyr Asp Thr Glu Thr Gly Pro Pro Thr Val Pro Phe Leu Thr Pro Pro Phe Val Ser Pro Asn Gly Phe Gln Glu Ser Pro Pro Gly Val Leu Ser 35 40 ~ 45 Leu Arg Leu Ser Glu Pro Leu Val Thr Ser Asn Gly Met Leu Ala Leu Lys Met Gly Asn Gly Leu Ser Leu Asp Glu Ala Gly Asn Leu Thr Ser 65 70 ' 75 80 Gln Asn Val Thr Thr Val Ser Pro Pro Leu Lys Lys Thr Lys Ser Asn Ile Asn Leu Glu Ile Ser Ala Pro Leu Thr Val Thr Ser Glu Ala Leu Thr Val Ala Ala Ala Ala Pro Leu Met Val Ala Gly Asn Thr Leu Thr Met Gln Ser Gln Ala Pro Leu Thr Val His Asp Ser Lys Leu Ser Ile Ala Thr Gln Gly Pro Leu Thr Val Ser Glu Gly Lys Leu Ala Leu Gln Thr Ser Gly Pro Leu Thr Thr Thr Asp Ser Ser Thr Leu Thr Ile Thr Ala Ser Pro Pro Leu Thr Thr Ala Thr Gly Ser Leu Gly Ile Asp Leu SUBSTITUTE SHEET (RULE 26) Lys Glu Pro Ile Tyr Thr Gln Asn Gly Lys Leu Gly Leu Lys Tyr Gly Ala Pro Leu His Val Thr Asp Asp Leu Asn Thr Leu Thr Val Ala Thr Gly Pro Gly Val Thr Ile Asn Asn Thr Ser Leu Gln Thr Lys Val Thr Gly Ala Leu Gly Phe Asp Ser Gln Gly Asn Met Gln Leu Asn Val Ala Gly Gly Leu Arg Ile Asp Ser Gln Asn Arg Arg Leu Ile Leu Asp Val Ser Tyr Pro Phe Asp Ala Gln Asn Gln Leu Asn Leu Arg Leu Gly Gln Gly Pro Leu Phe Ile Asn Ser Ala His Asn Leu Asp I1e Asn Tyr Asn Lys Gly Leu Tyr Leu Phe Thr Ala Ser Asn Asn Ser Lys Lys Leu Glu Va1 Asn Leu Ser Thr Ala Lys Gly Leu Met Phe Asp Ala Thr Ala Ile Ala Ile Asn Ala Gly Asp Gly Leu Glu Phe Gly Ser Pro Asn Ala Pro Asn Thr Asn Pro Leu Lys Thr Lys Ile Gly His Gly Leu Glu Phe Asp Ser Asn Lys Ala Met Val Pro Lys Leu Gly Thr Gly Leu Ser Phe Asp Ser Thr Gly Ala Ile Thr Val Gly Asn Lys Asn Asn Asp Lys Leu Thr Leu Trp Thr Thr PY~o Ala Pro Glu Ala Asn Cys Arg Leu Asn Ala Glu Lys Asp Ala Lys Leu Thr Leu Val Leu Thr Lys Cys Gly Ser Gln Ile Leu Ala Thr Val Ser Val Leu Ala Gly Ser Leu Ala Pro Ile Ser Gly SUBSTITUTE SHEET (RULE 26) Thr Val Gln Ser Ala His Leu Ile Ile Arg Phe Asp Glu Asn Gly Val Leu Leu Asn Asn Ser Phe Leu Asp Pro Glu Tyr Trp Asn Phe Arg Asn Gly Asp Leu Thr Glu Gly Thr Ala Tyr Thr Asn Ala Val Gly Phe Met Pro Asn Leu Ser Ala Tyr Pro Lys Ser His Gly Lys Thr Ala Lys Ser Asn Ile Val Ser Gln Val Tyr Leu Asn Gly Asp Lys Thr Lys Pro Val Thr Leu Thr Ile Thr Leu Asn Gly Thr Gln Glu Thr Gly Asp Thr Thr 530 535 . 540 Pro Ser Ala Tyr Ser Met Ser Phe Ser Trp Asp Trp Ser Gly His Asn Tyr Ile Asn Glu Ile Phe Ala Thr Ser Ser Tyr Thr Phe Ser Tyr Ile Ala Gln Glu <210> 9 <211> 1743 <212> DNA
<213> Artificial Sequence <220>
<223> Codes for a mutated Human Adenovirus type 5 fiber protein. Nucle otides corresponding to amino acid 441(V) of wild-type fiber was deleted.
<220>
<222> CDS
<222> (1)..(1743) <223>
<400> 9 atgaagcgcgca agaccg tctgaagat accttcaac cccgtg tatcca 48 MetLysArgAla ArgPro SerGluAsp ThrPheAsn ProVal TyrPro 1 5 w 10 15 , tatgacacggaa accggt cctccaact gtgcctttt cttact cctccc 96 TyrAspThrGlu ThrGly ProProThr ValProPhe LeuThr ProPro tttgtatccccc aatggg tttcaagag agtccccct ggggta ctctct 144 PheValSerPro AsnGly PheGlnGlu SerProPro GlyVal LeuSer ttgcgcctatcc gaacct ctagttacc tccaatggc atgctt gcgctc 192 LeuArgLeuSer GluPro LeuValThr SerAsnGly MetLeu AlaLeu SUBSTITUTE SHEET (RULE 26) aaaatgggcaac ggcctc tctctggac gaggcc ggcaacctt acctcc 240 LysMetGlyAsn G1yLeu SerLeuAsp GluAla GlyAsnLeu ThrSer caaaatgtaacc actgtg agcccacct ctcaaa aaaaccaag tcaaac 288 GlnAsnValThr ThrVal SerProPro LeuLys LysThrLys SerAsn ataaacctggaa atatct gcacccctc acagtt acctcagaa gcccta 336 IleAsnLeuGlu I1eSer AlaProLeu ThrVal ThrSerGlu AlaLeu actgtggetgcc gccgca cctctaatg gtcgcg ggcaacaca ctcacc 384 ThrValAlaAla AlaAla ProLeuMet ValAla GlyAsnThr LeuThr atgcaatcacag gccccg ctaaccgtg cacgac tccaaactt agcatt 432 MetGlnSerGln AlaPro LeuThrVal HisAsp SerLysLeu SerIle gccacccaagga cccctc acagtgtca gaagga aagctagcc ctgcaa 480 AlaThrGlnGly ProLeu ThrValSer GluGly LysLeuAla LeuGln acatcaggcccc ctcacc accaccgat agcagt acccttact atcact 528 ThrSerGlyPro LeuThr ThrThrAsp SerSer ThrLeuThr IleThr gcctcaccccct ctaact actgccact ggtagc ttgggcatt gacttg 576 AlaSerProPro LeuThr ThrAlaThr GlySer LeuGlyIle AspLeu aaagagcccatt tataca caaaatgga aaacta ggactaaag tacggg 624 LysGluProIle TyrThr GlnAsnGly LysLeu GlyLeuLys TyrGly getcctttgcat gtaaca gacgaccta aacact ttgaccgta gcaact 672 AlaProLeuHis ValThr AspAspLeu AsnThr LeuThrVal AlaThr ggtccaggtgtg actatt aataatact tccttg caaactaaa gttact 720 GlyProGlyVal ThrIle AsnAsnThr SerLeu GlnThrLys ValThr ggagccttgggt tttgat tcacaaggc aatatg caacttaat gtagca 768 GlyAlaLeuGly PheAsp SerGlnGly AsnMet GlnLeuAsn ValAla ggaggactaagg attgat tctcaaaac agacgc cttatactt gatgtt 816 GlyGlyLeuArg IleAsp SerGlnAsn ArgArg LeuIleLeu AspVal agttatccgttt gatget caaaaccaa ctaaat ctaagacta ggacag 864 SerTyrProPhe AspAla GlnAsnGln LeuAsn LeuArgLeu GlyGln ggccctcttttt ataaac tcagcccac aacttg gatattaac tacaac 912 GlyProLeuPhe IleAsn SerAlaHis AsnLeu AspIleAsn TyrAsn aaaggcctttac ttgttt acagettca aacaat tccaaaaag cttgag 960 LysGlyLeuTyr LeuPhe ThrAlaSer AsnAsn SerLysLys LeuGlu gtt aac cta agc act gcc aag ggg ttg atg ttt gac get aca gcc ata 1008 SUBSTITUTE SHEET (RULE 26) Val Asn Leu Ser Thr Ala Lys Gly Leu Met Phe Asp Ala Thr Ala Ile gcc att aat gca gga gat ggg ctt gaa ttt ggt tca cct aat gca cca 1056 Ala Ile Asn Ala Gly Asp Gly Leu Glu Phe Gly Ser Pro Asn Ala Pro aac aca aat ccc ctc aaa aca aaa att ggc cat ggc cta gaa ttt gat 1104 Asn Thr Asn Pro Leu Lys Thr Lys Ile Gly His Gly Leu Glu Phe Asp tcaaac aagget atggttcct aaacta ggaactggc cttagtttt gac 1152 SerAsn LysAla MetValPro LysLeu GlyThrGly LeuSerPhe Asp agcaca ggtgcc attacagta ggaaac aaaaataat gataagcta act 1200 SerThr GlyAla IleThrVal GlyAsn LysAsnAsn AspLysLeu Thr ttgtgg accaca ccagetcca tctcct aactgtaga ctaaatgca gag 1248 LeuTrp ThrThr ProAlaPro SerPro AsnCysArg LeuAsnAla Glu 405. 410 415 aaagat getaaa ctcactttg gtctta acaaaatgt ggcagtcaa ata 1296 LysAsp AlaLys LeuThrLeu ValLeu ThrLysCys GlySerGln Ile cttget acagtt tcagttttg getaaa ggcagtttg getccaata tct 1344 LeuAla ThrVal SerValLeu AlaLys GlySerLeu AlaProIle Ser ggaacagttcaa agtget catcttatt ataagattt gacgaaaat gga 1392 GlyThrValGln SerAla HisLeuIle IleArgPhe AspGluAsn Gly gtgctactaaac aattcc ttcctggac ccagaatat tggaacttt aga 1440 ValLeuLeuAsn AsnSer PheLeuAsp ProGluTyr TrpAsnPhe Arg aatggagatctt actgaa ggcacagcc tatacaaac getgttgga ttt 1488 AsnGlyAspLeu ThrGlu GlyThrAla TyrThrAsn AlaValGly Phe atgcctaaccta tcaget tatccaaaa tetcacggt aaaactgcc aaa 1536 MetProAsnLeu SerAla TyrProLys SerHisGly LysThrAla Lys agtaacattgtc agtcaa gtttactta aacggagac aaaactaaa cct 1584 SerAsnIleVal SerGln ValTyrLeu AsnGlyAsp LysThrLys Pro gtaacactaacc attaca etaaacggt acacaggaa acaggagac aca 1632 ValThrLeuThr IleThr LeuAsnGly ThrGlnGlu ThrGlyAsp Thr actccaagtgca tactct atgtcattt tcatgggac tggtctggc cac 16'80 ThrProSerAla TyrSex MetSerPhe SerTrpAsp TrpSerGly His aactacattaat gaaata tttgccaca tcctcttac actttttca tac 1728 AsnTyrIleAsn GluIle PheAlaThr SerSerTyr ThrPheSer Tyr attgcccaagaa taa 1743 IleAlaGlnGlu SUBSTITUTE SHEET (RULE 26) <210> 10 <211> 580 <212> PRT
<213> Artificial Sequence <220>
<223> Codes for a mutated Human Adenovirus type 5 fiber protein. Nucle otides corresponding to amino acid 441(V) of wild-type fiber was deleted.
<400> 10 Met Lys Arg Ala Arg Pro Ser Glu Asp Thr Phe Asn Pro Val Tyr Pro Tyr Asp Thr Glu Thr Gly Pro Pro Thr Val Pro Phe Leu Thr Pro Pro Phe Val Ser Pro Asn Gly Phe Gln Glu,_Ser Pro Pro Gly Val Leu Ser Leu Arg Leu Ser Glu Pro Leu Val Thr Ser Asn Gly Met Leu Ala Leu Lys Met Gly Asn Gly Leu Ser Leu Asp Glu Ala Gly Asn Leu Thr Ser 65 70 75 80 , Gln Asn Val Thr Thr Val Ser Pro Pro Leu Lys Lys Thr Lys Ser Asn Ile Asn Leu Glu Ile Ser Ala Pro Leu Thr Val Thr Ser Glu Ala Leu Thr Val Ala Ala Ala Ala Pro Leu Met Val Ala Gly Asn Thr Leu Thr Met Gln Ser Gln Ala Pro Leu Thr Val His Asp Ser Lys Leu Ser Ile Ala Thr Gln Gly Pro Leu Thr Val Ser Glu Gly Lys Leu Ala Leu Gln Thr Ser Gly Pro Leu Thr Thr Thr Asp Ser Ser Thr Leu Thr Ile Thr Ala Ser Pro Pro Leu Thr Thr Ala Thr Gly Ser Leu Gly Ile Asp Leu Lys Glu Pro Ile Tyr Thr Gln Asn Gly Lys Leu Gly Leu Lys Tyr Gly Ala Pro Leu His Val Thr Asp Asp Leu Asn Thr Leu Thr Val Ala Thr SUBSTITUTE SHEET (RULE 26) Gly Pro Gly Val Thr Ile Asn Asn Thr Ser Leu Gln Thr Lys Val Thr Gly Ala Leu Gly Phe Asp Ser Gln Gly Asn Met Gln Leu Asn Val Ala Gly Gly Leu Arg Ile Asp Ser Gln Asn Arg Arg Leu Ile Leu Asp Val Ser Tyr Pro Phe Asp Ala Gln Asn Gln Leu Asn Leu Arg Leu Gly Gln Gly Pro Leu Phe Ile Asn Ser Ala His Asn Leu Asp Ile Asn Tyr Asn Lys Gly Leu Tyr Leu Phe Thr Ala Ser Asn Asn Ser Lys Lys Leu Glu 305 3l0 315 320 Val Asn Leu Ser Thr Ala Lys Gly Leu Met Phe Asp Ala Thr Ala Ile Ala Ile Asn A1a Gly Asp Gly Leu Glu Phe Gly Ser Pro Asn Ala Pro Asn Thr Asn Pro Leu Lys Thr Lys Ile Gly His Gly Leu Glu Phe Asp Ser Asn Lys Ala Met Val Pro Lys Leu Gly Thr Gly Leu Ser Phe Asp Ser Thr Gly Ala Ile Thr Val Gly Asn Lys Asn Asn Asp Lys Leu Thr Leu Trp Thr Thr Pro Ala Pro Ser Pro Asn Cys Arg Leu Asn Ala Glu Lys Asp Ala Lys Leu Thr Leu Val Leu Thr Lys Cys Gly Ser Gln Ile Leu Ala Thr Val Ser Val Leu Ala Lys Gly Ser Leu Ala Pro Ile Ser Gly Thr Val Gln Ser Ala His Leu Ile Ile Arg Phe Asp Glu Asn Gly Val Leu Leu Asn Asn Ser Phe Leu Asp Pro Glu Tyr Trp Asn Phe Arg Asn Gly Asp Leu Thr Glu Gly Thr Ala Tyr Thr Asn Ala Val Gly Phe SUBSTITUTE SHEET (RULE 26) Met Pro Asn Leu Ser Ala Tyr Pro Lys Ser His Gly Lys Thr Ala Lys Ser Asn Ile Val Ser Gln Val Tyr Leu Asn Gly Asp Lys Thr Lys Pro Va1 Thr Leu Thr Ile Thr Leu Asn Gly Thr G1n Glu Thr G1y Asp Thr Thr Pro Ser Ala Tyr Ser Met Ser Phe Ser Trp Asp Trp Ser Gly His Asn Tyr Ile Asn Glu Ile Phe Ala Thr Ser Ser Tyr Thr Phe Ser Tyr Ile Ala Gln Glu <210> 11 <211> 1743 <212>. DNA
<213. Artificial Sequence <220>
<223> Codes for a mutated Human Adenovirus type 5 fiber protein. Nucle otides corresponding to amino acid 442(x) of wild-type fiber was deleted.
<220>
<221> CDS
<222> (1)..(1743) <223>
<400> 11 atgaagcgcgca agaccg tctgaagat accttcaac cccgtgtat cca 48 MetLysArgAla ArgPro SerGluAsp ThrPheAsn ProValTyr Pro tatgacacggaa accggt cctccaact gtgcctttt cttactcct ccc 96 TyrAspThrGlu ThrGly ProProThr ValProPhe LeuThrPro Pro tttgtatccccc aatggg tttcaagag agtccccct ggggtactc tct 144 PheValSerPro AsnGly PheGlnGlu SerProPro GlyValLeu Ser ttgcgcctatcc gaacct ctagttacc tccaatggc atgcttgcg ctc 192 LeuArgLeuSer GluPro LeuValThr SerAsnGly MetLeuAla Leu aaaatgggcaac ggcctc tctctggac gaggccggc aaccttacc tcc 240 LysMetGlyAsn GlyLeu SerLeuAsp GluAlaGly AsnLeuThr Ser caaaatgtaacc actgtg agcccacct ctcaaaaaa accaagtca aac 288 GlnAsnValThr ThrVal SerProPro LeuLy5Lys ThrLysSer Asn SUBSTITUTE SHEET (RULE 26) ataaac ctggaa atatctgca cccctc acagttacc tcagaagcc cta 336 IleAsn LeuGlu IleSerAla ProLeu ThrValThr SerGluAla Leu actgtg getgcc gccgcacct ctaatg gtcgcgggc aacacactc acc 384 ThrVal AlaAla AlaAlaPro LeuMet ValAlaG1y AsnThrLeu Thr atgcaa tcacag gccccgcta accgtg cacgactcc aaacttagc att 432 MetGln SerGln AlaProLeu ThrVal HisAspSer LysLeuSer Ile gccacc caagga cccctcaca gtgtca gaaggaaag ctagccctg caa 480 AlaThr GlnGly ProLeuThr ValSer GluGlyLys LeuAlaLeu Gln acatca ggcccc ctcaccacc accgat agcagtacc cttactatc act 528 ThrSer GlyPro LeuThrThr ThrAsp SerSerThr LeuThrIle Thr gcctca ccccct ctaactact gccact ggtagcttg ggcattgac ttg 576 AlaSer ProPro LeuThrThr AlaThr GlySexLeu GlyIleAsp Leu aaagag cccatt tatacacaa aatgga aaactagga ctaaagtac ggg 624 LysGlu Pro21e TyrThrGln AsnGly LysLeuGly LeuLysTyr Gly getcct ttgcat gtaacagac gaccta aacactttg accgtagca act 672 AlaPro LeuHis Val,T3lrAsp AspLeu AsnThrLeu ThrValAla Thr ggtcca ggtgtg actattaat aatact tccttgcaa actaaagtt act 720 GlyPro GlyVal ThrIleAsn AsnThr SerLeuGln ThrLysVal Thr ggagcc ttgggt tttgattca caaggc aatatgcaa cttaatgta gca 768 G1yAla LeuGly PheAspSer GlnGly AsnMetGln LeuAsnVal Ala ggagga ctaagg attgattct caaaac agacgcctt atacttgat gtt 816 GlyGly LeuArg IleAspSer GlnAsn ArgArgLeu Ile-LeuAsp Val agttat ccgttt gatgetcaa aaccaa ctaaatcta agactagga cag 864 SerTyr ProPhe AspAlaGln AsnGln LeuAsnLeu ArgLeuGly Gln ggccct cttttt ataaactca gcccac aacttggat attaactac aac 912 GlyPro LeuPhe IleAsnSer AlaHis AsnLeuAsp IleAsnTyr Asn aaaggc ctttac ttgtttaca gettca aacaattcc aaaaagctt gag 960 LysG1y LeuTyr LeuPheThr AlaSer AsnAsnSer LysLysLeu Glu gttaac ctaagc actgccaag gggttg atgtttgac getacagcc ata 1008 ValAsn LeuSer ThrAlaLys GlyLeu MetPheAsp AlaThrAla Ile gccatt aatgca ggagatggg cttgaa tttggttca cctaatgca cca 1056 AlaIle AsnAla GlyAspGly LeuGlu PheGlySer ProAsnAla Pro aacaca aatccc ctcaaaaca aaaatt ggccatggc ctagaattt gat 1104 AsnThr AsnPro LeuLysThr LysIle GlyHisGly LeuGluPhe Asp SUBSTITUTE SHEET (RULE 26) tcaaacaag getatggttcct aaacta ggaact ggccttagt tttgac 1152 SerAsnLys AlaMetValPro LysLeu GlyThr GlyLeuSer PheAsp agcacaggt gccattacagta ggaaac aaaaat aatgataag ctaact 1200 SerThrGly AlaIleThrVal GlyAsn LysAsn AsnAspLys LeuThr ttgtggacc acaccagetcca tctcct aactgt agactaaat gcagag 1248 LeuTrpThr ThrProAlaPro SerPro AsnCys ArgLeuAsn AlaGlu aaagatget aaactcactttg gtctta acaaaa tgtggcagt caaata 1296 LysAspAla LysLeuThrLeu ValLeu ThrLys CysGlySer GlnIle cttgetaca gtttcagttttg getgtt ggcagt ttggetcca atatct 1344 LeuAlaThr ValSerValLeu AlaVal GlySer LeuAlaPro IleSer ggaacagtt caaagtgetcat cttatt ataaga tttgacgaa aatgga 1392 GlyThrVal GlnSerAlaHis LeuIle IleArg PheAspGlu AsnGly gtgctacta aacaattccttc ctggac ccagaa tattggaac tttaga 1440 ValLeuLeu AsnAsnSerPhe LeuAsp ProGlu TyrTrpAsn PheArg aatggagat cttactgaaggc acagcc 'tataca aacgetgtt ggattt 1488 AsnGlyAsp LeuThrGluGly ThrAla TyrThr AsnAlaVal GlyPhe atgcctaac ctatcagettat ccaaaa tctcac ggtaaaact gccaaa 1536 MetProAsn LeuSerAlaTyr ProLys SerHis GlyLysThr AlaLys agtaacatt gtcagtcaagtt tactta aacgga gacaaaact aaacct 1584 SerAsnIle ValSerGlnVal TyrLeu AsnGly AspLysThr LysPro gtaacacta accattacacta aacggt acacag gaaacagga gacaca 1632 ~

ValThrLeu ThrIleThrLeu AsnGly ThrGln GluThrGly AspThr actccaagt gcatactctatg tcattt tcatgg gactggtct ggccac 1680 ThrProSer AlaTyrSerMet SerPhe SerTrp AspTrpSer GlyHis aactacatt aatgaaatattt gccaca tcctct tacactttt tcatac 1728 AsnTyrIle AsnGluIlePhe AlaThr SerSer TyrThrPhe SerTyr attgcccaa gaataa 1743 IleAlaGln Glu <210> 12 <211> 580 <212> PRT

<213>
Artificial Sequence <220>
<223> Codes for a mutated Human Adenovirus type 5 fiber protein. Nucle otides corresponding to amino acid 442(x) of wild-type fiber was SUBSTITUTE SHEET (RULE 26) deleted.
<400> 12 Met Lys Arg Ala Arg Pro Ser Glu Asp Thr Phe Asn Pro Val Tyr Pro Tyr Asp Thr Glu Thr Gly Pro Pro Thr Val Pro Phe Leu Thr Pro Pro Phe Val Ser Pro Asn Gly Phe Gln Glu Ser Pro Pro Gly Val Leu Ser Leu Arg Leu Ser Glu Pro Leu Val Thr Ser Asn Gly Met Leu Ala Leu Lys Met Gly Asn Gly Leu Ser Leu Asp Glu Ala Gly Asn Leu Thr Ser Gln Asn Val Thr Thr Val Ser Pro Pro Leu Lys Lys Thr Lys Ser Asn Ile Asn Leu Glu Ile Ser Ala Pro Leu Thr Val Thr Ser Glu Ala Leu Thr Val Ala Ala Ala Ala Pro Leu Met Val Ala Gly Asn Thr Leu Thr Met Gln Ser G1n Ala Pro Leu Thr Val His Asp Ser Lys Leu Ser Ile Ala Thr Gln Gly Pro Leu Thr Val Ser Glu Gly Lys Leu Ala Leu Gln 145 _150 _ 155 ,_ 160 Thr Ser Gly Pro Leu Thr Thr Thr Asp Ser Ser Thr Leu Thr Ile Thr Ala Ser Pro Pro Leu Thr Thr Ala Thr Gly Ser Leu Gly Ile Asp Leu Lys Glu Pro Ile Tyr Thr Gln Asn Gly Lys Leu Gly Leu Lys Tyr Gly Ala Pro Leu His Val Thr Asp Asp Leu Asn Thr Leu Thr Val Ala Thr Gly Pro Gly Val Thr Ile Asn Asn Thr Ser Leu Gln Thr Lys Val Thr Gly Ala Leu Gly Phe Asp Ser Gln Gly Asn Met Gln Leu Asn Val Ala SUBSTITUTE SHEET (RULE 26) Gly Gly Leu Arg Ile Asp Ser Gln Asn Arg Arg Leu Ile Leu Asp Val Ser Tyr Pro Phe Asp Ala Gln Asn Gln Leu Asn Leu Arg Leu Gly Gln Gly Pro Leu Phe Ile Asn Ser Ala His Asn Leu Asp Ile Asn Tyr Asn Lys Gly Leu Tyr Leu Phe Thr Ala Ser Asn Asn Ser Lys Lys Leu Glu Val Asn Leu Ser Thr Ala Lys Gly Leu Met Phe Asp Ala Thr Ala Ile Ala Ile Asn Ala Gly Asp Gly Leu Glu Phe. Gly Ser Pro Asn Ala Pro 340 345 35b Asn Thr Asn Pro Leu Lys Thr Lys Ile Gly His Gly Leu Glu Phe Asp Ser Asn Lys Ala Met Val Pro Lys Leu Gly Thr Gly Leu Ser Phe Asp 370 ~ 375 380 Ser Thr Gly Ala Ile Thr Val Gly Asn Lys Asn Asn Asp Lys Leu Thr 3g5 390 395 400 Leu Trp Thr Thr Pro Ala Pro Ser Pro Asn Cys Arg Leu Asn Ala Glu Lys Asp Ala Lys Leu Thr Leu Val Leu Thr Lys Cys Gly Ser Gln Ile 420 425 '~ 430 Leu Ala Thr Val Ser Val Leu Ala Val Gly Ser Leu Ala Pro Ile Ser Gly Thr Val Gln Ser Ala His Leu Ile Ile Arg Phe Asp Glu Asn Gly Val Leu Leu Asn Asn Ser Phe Leu Asp Pro Glu Tyr Trp Asn Phe Arg Asn Gly Asp Leu Thr Glu Gly Thr Ala Tyr Thr Asn Ala Val Gly Phe Met Pro Asn Leu Ser Ala Tyr Pro Lys Ser His Gly Lys Thr Ala Lys Ser Asn Ile Val Ser Gln Val Tyr Leu Asn Gly Asp Lys Thr Lys Pro SUBSTITUTE SHEET (RULE 26) Val Thr Leu Thr Ile Thr Leu Asn Gly Thr Gln Glu Thr Gly Asp Thr Thr Pro Ser Ala Tyr Ser Met Ser Phe Ser Trp Asp Trp Ser Gly His Asn Tyr Ile Asn Glu Ile Phe Ala Thr Ser Ser Tyr Thr Phe Ser Tyr Ile Ala Gln Glu <210> 13 <211> 1746 <212> DNA
<213> Artificial Sequence _, <220>
<223> Codes for a mutated Human Adenovirus type 5 fiber protein.
<220>
<221> CDS
<222> (1)..(1746) <223>
<220>
<221> mutation <222> (1321)..(1326) <223>
<400>

atgaagcgc gcaagaccg tctgaagat accttc aaccccgtg tatcca 48 MetLysArg AlaArgPro SerGluAsp ThrPhe AsnProVal TyrPro tatgacacg gaaaccggt cctccaact gtgcct tttcttact cctccc 96 TyrAspThr GluThrGly ProProThr ValPro PheLeuThr ProPro tttgtatcc cccaatggg tttcaagag agtccc cctggggta ctctct 144 PheValSer ProAsnGly PheGlnGlu SerPro ProGlyVal LeuSer ttgcgccta tccgaacct ctagttacc tccaat ggcatgctt gcgctc 192 LeuArgLeu SerGluPro LeuValThr SerAsn GlyMetLeu AlaLeu aaaatgggc aacggcctc tctctggac gaggcc ggcaacctt acctcc 240 LysMetGly AsnGlyLeu SerLeuAsp GluAla GlyAsnLeu ThrSer caaaatgta accactgtg agcccacct ctcaaa aaaaccaag tcaaac 288 GlnAsnVal ThrThrVal SerProPro LeuLys LysThrLys SerAsn g5 90 95 ataaacctg gaaatatct gcacccctc acagtt acctcagaa gcccta 336 IleAsnLeu GluIleSer AlaProLeu ThrVal ThrSerGlu AlaLeu SUBSTITUTE SHEET (RULE 26) act gtggetgcc gccgca cctctaatg gtcgcg ggcaacaca ctcacc 384 Thr ValAlaAla AlaAla ProLeuMet ValAla GlyAsnThr LeuThr atg caatcacag gccccg ctaaccgtg cacgac tccaaactt agcatt 432 Met GlnSerGln AlaPro LeuThrVal HisAsp SerLysLeu SerIle gcc acccaagga cccctc acagtgtca gaagga aagctagCC Ctgcaa 480 Ala ThrGlnGly ProLeu ThrValSer GluGly LysLeuAla LeuGln aca tcaggCCCC CtCaCC aCCaCCgat agcagt acccttact atcact 528 Thr SerGlyPro LeuThr ThrThrAsp SerSer ThrLeuThr IleThr gcc tcaccccct ctaact actgccact ggtagc ttgggcatt gacttg 576 Ala SerProPro LeuThr ThrAlaThr GlySer LeuGlyIle AspLeu aaa gagcccatt tataca caaaatgga aaacta ggactaaag tacggg 624 Lys GluProIle TyrT,hrGlnAsnGly LysLeu GlyLeuLys T_yrGly get cctttgcat gtaaca gacgaccta aacact ttgaccgta gcaact 672 Ala ProLeuHis ValThr AspAspLeu AsnThr LeuThrVal AlaThr ggt ccaggtgtg actatt aataatact tccttg caaactaaa gttact 720 Gly Pr.~?GlyVal ThrIle AsnAsnThr SerLeu GlnThrLys Va.lThr gga gccttgggt tttgat tcacaaggc aatatg caacttaat gtagca 768 Gly AlaLeuGly PheAsp SerGlnGly AsnMet GlnLeuAsn ValAla gga ggactaagg attgat tctcaaaac agacgc cttatactt gatgtt 816 Gly GlyLeuArg IleAsp SerGlnAsn ArgArg LeuIleLeu AspVal 260 265 270 ' agt tatccgttt gatget caaaaccaa ctaaat ctaagacta ggacag 864 Ser.TyrProPhe AspAla GlnAsnGln LeuAsn LeuArgLeu GlyGln ggc cctcttttt ataaac tcagcccac aacttg gatattaac tacaac 912 Gly ProLeuPhe IleAsn SerAlaHis AsnLeu AspIleAsn TyrAsn aaa ggcctttac ttgttt acagettca aacaat tccaaaaag cttgag 960 Lys GlyLeuTyr LeuPhe ThrAlaSer AsnAsn SerLysLys LeuGlu gtt aacctaagc actgcc aaggggttg atgttt gacgetaca gccata 1008 Val AsnLeuSer ThrAla LysGlyLeu MetPhe AspAlaThr AlaIle gcc attaatgca ggagat gggcttgaa tttggt tcacctaat gcacca 1056 Ala IleAsnAla GlyAsp GlyLeuGlu PheGly SerProAsn AlaPro aac acaaatccc ctcaaa acaaaaatt ggccat ggcctagaa tttgat 1104 Asn ThrAsnPro LeuLys ThrLysIle GlyHis GlyLeuGlu PheAsp tca aacaagget atggtt cctaaacta ggaact ggccttagt tttgac 1152 Ser AsnLysAla MetVal ProLysLeu GlyThr GlyLeuSer PheAsp 3~
SUBSTITUTE SHEET (RULE 26) agcacaggtgcc attaca gtaggaaac aaaaat aatgataag ctaact 1200 SerThrGIyAla IleThr ValGIyAsn LysAsn AsnAspLys LeuThr ttgtggaccaca ecaget ccatctect aactgt agactaaat gcagag 1248 LeuTrpThrThr ProAla ProSerPro AsnCys ArgLeuAsn AlaGlu aaagatgetaaa ctcact ttggtetta acaaaa tgtggcagt caaata 1296 LysAspAlaLys LeuThr LeuValLeu ThrLys CysGlySer GlnIle cttgetaca.gtttcagtt ttggetget gcaggc agtttgget ccaata 1344 LeuAlaThrVal SerVal LeuAlaAla AlaGly SerLeuAla ProIle tctggaacagtt caaagt getcatctt attata agatttgac gaaaat 1392 SerGlyThrVal GlnSer AlaHisLeu IleIle ArgPheAsp GluAsn ggagtgctacta aacaat tccttcctg gaccca gaatat.tgg .aacttt 1440 GlyValLeuLeu AsnAsn SerPheLeu AspPro GluTyrTrp AsnPhe agaaatggagat cttact gaaggcaca gcctat acaaacget gttgga 1488 ArgAsnGlyAsp LeuThr GluGlyThr AlaTyr ThrAsnAla ValGly tttatgcctaac ctatca gettatcea aaatct caeggtaaa actgcc 1536 PheMetProAsn LeuSer AlaTyrPro LysSer HisGlyLys ThrAla aaaagtaacatt gtcagt caagtttac ttaaac ggagacaaa actaaa 1584 LysSerAsnIle ValSer GlnValTyr LeuAsn GlyAspLys ThrLys cctgtaacacta accatt acactaaac ggtaca caggaaaca ggagac 1632 ProValThrLeu ThrIle ThrLeuAsn GlyThr GlnGluThr GlyAsp acaactccaagt gcatac tctatgtca ttttca tgggactgg tctggc 1680 ~

ThrThrProSer AlaTyr SerMetSer PheSer TrpAspTrp SerGly cacaactacatt aatgaa atatttgcc acatcc tcttacact ttttca 1728 HisAsnTyrIle AsnGlu IlePheAla ThrSer SerTyrThr PheSer tacattgcccaa gaataa 1746 TyrIleAlaGln Glu <210> 14 <211> 581 <212> PRT
<213> Artificial Sequence <220>
<223> Codes for a mutated Human Adenovirus type 5 fiber protein.
<400> 14 Met Lys Arg Ala Arg Pro Ser Glu Asp Thr Phe Asn Pro Val Tyr Pro l 5 10 15 SUBSTITUTE SHEET (RULE 26) Tyr Asp Thr G1u Thr Gly Pro Pro Thr Val Pro Phe Leu Thr Pro Pro Phe Val Ser Pro Asn Gly Phe Gln Glu Ser Pro Pro Gly Val Leu Ser Leu Arg Leu Ser Glu Pro Leu Val Thr Ser Asn Gly Met Leu Ala Leu Lys Met Gly Asn Gly Leu Ser Leu Asp Glu Ala Gly Asn Leu Thr Ser Gln Asn Val Thr Thr Val Ser Pro Pro Leu Lys Lys Thr Lys Ser Asn Ile Asn Leu Glu_Ile_,Ser Ala Pro Leu Thr Val Thr Ser Glu Ala Leu 100 ' 105 110 Thr Val Ala Ala Ala Ala Pro Leu Met Val Ala Gly Asn Thr Leu Thr Met Gln Ser Gln Ala Pro Leu Thr Val His Asp Ser Lys Leu Ser Ile 130 135 , . 140 Ala Thr Gln Gly Pro Leu Thr Val Ser Glu Gly Lys Leu Ala Leu Gln Thr Ser Gly Pro Leu Thr Thr Thr Asp Ser Ser Thr Leu Thr Ile Thr Ala Ser Pro Pro Leu Thr Thr Ala Thr Gly Ser Leu_Gly Ile Asp Leu 180 ~ 185 190 Lys Glu Pro Ile Tyr Thr Gln Asn Gly Lys Leu Gly Leu Lys Tyr Gly Ala Pro Leu His Val Thr Asp Asp Leu Asn Thr Leu Thr Val Ala Thr Gly Pro Gly Val Thr Ile Asn Asn Thr Ser Leu Gln Thr Lys Val Thr Gly Ala Leu Gly Phe Asp Ser Gln Gly Asn Met Gln Leu Asn Val Ala Gly Gly Leu Arg Ile Asp Ser Gln Asn Arg Arg Leu Ile Leu Asp Val Ser Tyr Pro Phe Asp Ala Gln Asn Gln Leu Asn Leu Arg Leu Gly Gln SUBSTITUTE SHEET (RULE 26) Gly Pro Leu Phe Ile Asn Ser Ala His Asn Leu Asp Ile Asn Tyr Asn Lys Gly Leu Tyr Leu Phe Thr Ala Ser Asn Asn Ser Lys Lys Leu Glu 305 310 315 , 320 Val Asn Leu Ser Thr Ala Lys Gly Leu Met Phe Asp Ala Thr Ala Tle Ala Ile Asn Ala Gly Asp Gly Leu Glu Phe Gly Ser Pro Asn Ala Pro Asn Thr Asn Pro Leu Lys Thr Lys Ile Gly His Gly Leu Glu Phe Asp Ser Asn Lys Ala Met Val Pro Lys Leu~Gly Thr Gly Leu Ser Phe Asp Ser Thr Gly Ala Ile Thr Val Gly Asn Lys Asn Asn Asp Lys Leu Thr Leu,.Trp Thr Thr Pro Ala Pro Ser Pro Asn Cys Arg Leu Asn,~~la Glu , Lys Asp Ala Lys Leu Thr Leu Val Leu Thr Lys Cys Gly Ser Gln Ile Leu Ala Thr Val Ser Val Leu Ala Ala Ala Gly Ser Leu Ala Pro Ile Ser Gly Thr Val Gln Ser Ala His Leu Ile Ile Arg Phe Asp Glu Asn Gly Val Leu Leu Asn Asn Ser Phe Leu Asp Pro Glu Tyr Trp Asn Phe Arg Asn Gly Asp Leu Thr Glu Gly Thr Ala Tyr Thr Asn Ala Val Gly Phe Met Pro Asn Leu Ser Ala Tyr Pro Lys Ser His Gly Lys Thr Ala Lys Ser Asn Ile Val Ser Gln Val Tyr Leu Asn Gly Asp Lys Thr Lys Pro Val Thr Leu Thr Ile Thr Leu Asn Gly Thr Gln Glu Thr Gly Asp Thr Thr Pro Ser Ala Tyr Ser Met Ser Phe Ser Trp Asp Trp Ser Gly SUBSTITUTE SHEET (RULE 26) His Asn Tyr Ile Asn Glu Ile Phe Ala Thr Ser Ser Tyr Thr Phe Ser Tyr Ile Ala Gln Glu <210> 15 <211> 1746 <212> DNA
<213> Artificial Sequence <220>
<223> Codes for a mutated Human Adenovirus type 5 fiber protein.
<220>
<221> CDS
.<.2.22> (1) ... (1746) <223>
<220>
<221> mutation <222> (1378)..(1380) <223>
<400> 15 atg aagcgcgca agaccg tctgaagat accttcaac cccgtg tatcca 48 Met LysArgAla ArgPro SerGluAsp ThrPheAsn ProVal TyrPro tat gacacggaa accggt cctccaact gtgcctttt cttact cctccc 96 Tyr AspThrGlu ThrGly ProProThr ValProPhe LeuThr ProPro ttt gtatccccc aatggg tttcaagag agtccccct ggggta ctctct 144 Phe ValSerPro AsnGly PheGlnGlu SerProPro GlyVal LeuSer 35 ~ 40 ~ ' 45 ttg cgcctatcc gaacct ctagttacc tccaatggc atgctt gcgctc 192 Leu ArgLeuSer GluPro LeuValThr SerAsnGly MetLeu AlaLeu aaa atgggcaac ggcctc tctctggac gaggccggc aacctt acctcc 240 Lys MetGlyAsn GlyLeu SerLeuAsp GluAlaGly AsnLeu ThrSer caa aatgtaacc actgtg agcccacct ctcaaaaaa accaag tcaaac 288 Gln AsnValThr ThrVal SerProPro LeuLysLys ThrLys SerAsn ata aacctggaa atatct gcacccctc acagttacc tcagaa gcccta 336 Ile AsnLeuGlu IleSer AlaProLeu ThrValThr SerGlu AlaLeu act gtggetgcc gccgca cctctaatg gtcgcgggc aacaca ctcacc 384 Thr ValAlaAla AlaAla ProLeuMet ValAlaGly AsnThr LeuThr atg caatcacag gccccg ctaaccgtg cacgactcc aaactt agcatt 432 Met GlnSerGln AlaPro LeuThrVal HisAspSer LysLeu SerIle SUBSTITUTE SHEET (RULE 26) gccacccaa ggacccctc acagtgtca gaaggaaag ctagcc ctgcaa 480 AlaThrGln GlyProLeu ThrValSer GluGlyLys LeuAla LeuGln acatcaggc cccctcacc accaccgat agcagtacc cttact atcact 528 ThrSerGly ProLeuThr ThrThrAsp SerSerThr LeuThr IleThr gcctcaccc cctctaact actgccact ggtagcttg ggcatt gacttg 576 AlaSerPro ProLeuThr ThrAlaThr GlySerLeu GlyIle AspLeu aaagagccc atttataca caaaatgga aaactagga ctaaag tacggg 624 LysGluPro IleTyrThr GlnAsnGly LysLeuGly LeuLys TyrGly getcctttg catgtaaca gacgaccta aacactttg accgta gcaact 672 AlaProLeu HisValThr AspAspLeu AsnThrLeu ThrVal AlaThr ggt.ccaggt gtgact,att aataatact tccttgcaa actaaa gttact 72,0 ~ ..

GlyProGly ValThrIle AsnAsnThr SerLeuGln ThrLys V'alThr 225 ' 230 235 240 ggagccttg ggttttgat tcacaaggc aatatgcaa cttaat gtagca 768 GlyAlaLeu GlyPheAsp SerGlnGly AsnMetGln LeuAsn ValAla ggaggacta aggattgat tctcaaaac agacgcctt atactt gatgtt 816 GlyGlyLeu ArgIleAsp SerGlnAsn ArgArgLeu IleLeu AspVal agttatccg tttgatget caaaaccaa ctaaatcta agacta ggacag 864 SerTyrPro PheAspAla GlnAsnGln LeuAsnLeu ArgLeu GlyGln ggccctctt tttataaac tcagcccac aacttggat attaac tacaac 912 GlyProLeu PheIleAsn SerAlaHis AsnLeuAsp IleAsn TyrAsn aaaggcctt tacttgttt acagettea aacaattcc aaaaag cttgag 960,.

LysGlyLeu TyrLeuPhe ThrAlaSer AsnAsnSer LysLys LeuGlu gttaaccta agcactgcc aaggggttg atgtttgac getaca gccata 1008 ValAsnLeu SerThrAla LysGlyLeu MetPheAsp AlaThr AlaIle gccattaat gcaggagat gggcttgaa tttggttca cctaat gcacca 1056 AlaIleAsn AlaGlyAsp GlyLeuGlu PheGlySer ProAsn AlaPro aacacaaat cccctcaaa acaaaaatt ggccatggc ctagaa tttgat 1104 AsnThrAsn ProLeuLys ThrLysIle GlyHisGly LeuGlu PheAsp tcaaacaag getatggtt cctaaacta ggaactggc cttagt tttgac 1152 SerAsnLys AlaMetVal ProLysLeu GlyThrGly LeuSer PheAsp agcacaggt gccattaca gtaggaaac aaaaataat gataag ctaact 1200 SerThrGly AlaIleThr ValGlyAsn LysAsnAsn AspLys LeuThr ttgtggacc acaccaget ccatctcct aactgtaga ctaaat gcagag 1248 LeuTrpThr ThrProAla ProSerPro AsnCysArg LeuAsn AlaGlu SUBSTITUTE SHEET (RULE 26) aaagat getaaa ctcactttg gtctta acaaaatgt ggcagtcaa ata 1296 LysAsp AlaLys LeuThrLeu ValLeu ThrLysCys GlySerGln Ile cttget acagtt tcagttttg getgtt aaaggcagt ttggetcca ata 1344 LeuAla ThrVal SerValLeu AlaVal LysGlySer LeuAlaPro Ile tctgga acagtt caaagtget catctt attatagaa ttcgacgaa aat 1392 SerGly ThrVal GlnSerAla HisLeu IleIleGlu PheAspGlu Asn ggagtg ctacta aacaattcc ttcctg gacccagaa tattggaac ttt 1440 GlyVal LeuLeu AsnAsnSer PheLeu AspProGlu TyrTrpAsn Phe agaaat ggagat cttactgaa ggcaca gcctataca aacgetgtt gga 1488 ArgAsn GlyAsp LeuThrGlu GlyThr AlaTyrThr AsnAlaVal Gly tttatg cctaac ctatca ~gcttatcca aaa~tctcac ggtaaa actgcc 153 6 PheMet ProAsn LeuSer AlaTyrPro LysSerHis GlyLys ThrAla aaaagt aacatt gtcagt caagtttac ttaaacgga gacaaa actaaa 1584 LysSer AsnIle ValSer GlnValTyr LeuAsnGly AspLys ThrLys cctgta ~ac:acta accatt acactaaac ggtacacag gaaaca gga:gac 1632 ProVal ThrLeu ThrIle ThrLeuAsn GlyThrGln GluThr GlyAsp acaact ccaagt gcatac tctatgtca ttttcatgg gactgg tctggc 1680 ThrThr ProSer AlaTyr SerMetSer PheSerTrp AspTrp SerGly cacaac tacatt aatgaa atatttgcc acatcctct tacact ttttca 1728 HisAsn TyrIle AsnGlu IlePheAla ThrSerSer TyrThr PheSer tacatt gcccaa gaataa 1746 TyrIle AlaGln Glu <210> 16 <211> 581 <212> PRT
<213> Artificial Sequence <220>
<223> Codes for a mutated Human Adenovirus type 5 fiber protein.
<400> 16 Met Lys Arg Ala Arg Pro Ser Glu Asp Thr Phe Asn Pro Val Tyr Pro Tyr Asp Thr Glu Thr Gly Pro Pro Thr Val Pro Phe Leu Thr Pro Pro Phe Val Ser Pro Asn Gly Phe Gln Glu Ser Pro Pro Gly Val Leu Ser SUBSTITUTE SHEET (RULE 26) Leu Arg Leu Ser Glu Pro Leu Val Thr Ser Asn Gly Met Leu Ala Leu Lys Met Gly Asn Gly Leu Ser Leu Asp Glu Ala Gly Asn Leu Thr Ser Gln Asn Val Thr Thr Val Ser Pro Pro Leu Lys Lys Thr Lys Ser Asn Ile Asn Leu Glu Ile Ser Ala Pro Leu Thr Val Thr Ser Glu Ala Leu Thr Val Ala Ala Ala Ala Pro Leu Met Val Ala Gly Asn Thr Leu Thr Met Gln Ser Gln Ala Pro Leu Thr Val His Asp Ser Lys Leu Ser Ile Ala Thr Gln Gly Pro Leu Thr Val Ser Glu Gly Lys Leu Ala Leu Gln Thr Ser Gly Pro Leu Thr Thr°Thr Asp Ser Ser Thr Leu Thr Ile Thr , Ala Ser Pro Pro Leu Thr Thr Ala Thr Gly Ser Leu Gly Ile Asp Leu Lys Glu Pro Ile Tyr Thr Gln Asn Gly Lys Leu Gly Leu Lys Tyr Gly Ala Pro Leu His Val Thr Asp Asp Leu Asn Thr Leu Thr Val Ala Thr Gly Pro Gly Val Thr Ile Asn Asn Thr Ser Leu Gln Thr Lys Val Thr 225 230 ~ 235 240 Gly Ala Leu Gly Phe Asp Ser Gln Gly Asn Met Gln Leu Asn Val Ala Gly Gly Leu Arg Ile Asp Ser Gln Asn Arg Arg Leu Ile Leu Asp Val Ser Tyr Pro Phe Asp Ala Gln Asn Gln Leu Asn Leu Arg Leu Gly Gln Gly Pro Leu Phe Ile Asn Ser Ala His Asn Leu Asp Ile Asn Tyr Asn Lys Gly Leu Tyr Leu Phe Thr Ala Ser Asn Asn Ser Lys Lys Leu Glu SUBSTITUTE SHEET (RULE 26) Val Asn Leu Ser Thr Ala Lys Gly Leu Met Phe Asp Ala Thr Ala Ile Ala Ile Asn Ala Gly Asp Gly Leu Glu Phe Gly Ser Pro Asn Ala Pro Asn Thr Asn Pro Leu Lys Thr Lys Ile Gly His Gly Leu Glu Phe Asp Ser Asn Lys Ala Met Val Pro Lys Leu Gly Thr Gly Leu Ser Phe Asp Ser Thr Gly Ala Ile Thr Val Gly Asn Lys Asn Asn Asp Lys Leu Thr Leu Trp Thr Thr Pro Ala Pro Ser Pro Asn Cys Arg Leu Asn Ala Glu Lys Asp Ala Lys Leu Thr Leu Val Leu Thr Lys Cys Gly Ser Gln Ile Leu Ala Thr Val Ser Val Leu Ala Val Lys Gly Ser Leu Ala Pro Ile Ser Gly Thr Val Gln Ser Ala His Leu Ile Ile Glu Phe Asp Glu Asn Gly Val Leu Leu Asn Asn Ser Phe Leu Asp Pro Glu Tyr Trp Asn Phe Arg Asn Gly Asp Leu Thr Glu Gly Thr Ala Tyr Thr Asn Ala Val Gly Phe Met Pro Asn Leu Ser Ala Tyr Pro Lys Ser His Gly Lys Thr Ala Lys Ser Asn Ile Val Ser Gln Val Tyr Leu Asn Gly Asp Lys Thr Lys Pro Val Thr Leu Thr Ile Thr Leu Asn Gly Thr Gln Glu Thr Gly Asp Thr Thr Pro Ser Ala Tyr Ser Met Ser Phe Ser Trp Asp Trp Ser Gly His Asn Tyr Ile Asn Glu Ile Phe Ala Thr Ser Ser Tyr Thr Phe Ser SUBSTITUTE SHEET (RULE 26) Tyr Ile Ala Gln Glu <210> 17 <211> 1740 <212> DNA
<213> Artificial Sequence <220>
<223> Codes for a mutated Human Adenovirus type 5 fiber protein. Nucle otides corresponding to amino acids 509(G) & 510(x) of wild-type fiber. were deleted.
<220>
<221> CDS
<222> (1)..(1740) <223>
<400> 17 atgaagcgcgca agaccg tctgaagat accttcaac cccgtgtat cca 48 MetLysArgAla ArgPro SerGluAsp ThrPheAsn ProValTyr Pro tatgacacggaa accggt cctccaact gtgcctttt cttactcct ccc 96 TyrAspThrGlu ThrGly ProProThr ValProPhe LeuThrPro Pro tttgtatccccc aatggg tttcaagag agtCCCCCt ggggtaCtC tct 144 PheValSerPro AsnGly PheGlnGlu SerProPro GlyValLeu Sea.:

ttgcgcctatcc gaacct ctagttacc tccaatggc atgcttgcg ctc 192 LeuArgLeuSer GluPro LeuValThr SerAsnGly MetLeuAla Leu ~

aaaatgggcaac ggcctc tctctggac gaggccggc aaccttacc tcc 240 LysMetGlyAsn GlyLeu SerLeuAsp GluAlaGly AsnLeuThr Ser caaaatgtaacc actgtg agcccacct ctcaaaaaa accaagtca aac 288 GlnAsnValThr ThrVal SerProPro LeuLysLys ThrLysSer Asn ataaacctggaa atatct gcacccctc acagttacc tcagaagcc cta 336 IleAsnLeuGlu IleSer AlaProLeu ThrValThr SerGluAla Leu actgtggetgcc gccgca cctctaatg gtcgcgggc aacacactc acc 384 ThrValAlaAla AlaAla ProLeuMet ValAlaGly AsnThrLeu Thr atgcaatcacag gccccg ctaaccgtg cacgactcc aaacttagc att 432 MetGlnSerGln AlaPro LeuThrVal HisAspSer LysLeuSer Ile 130 13'5 140 gccacccaagga cccctc acagtgtca gaaggaaag ctagccctg caa 480 AlaThrGlnGly ProLeu ThrValSer GluGlyLys LeuAlaLeu Gln acatcaggCCCC CtCaCC aCCaCCgat agcagtacc cttactatc act 528 ThrSerGlyPro LeuThr ThrThrAsp SerSerThr LeuThrIle Thr gcctcaccccct ctaact actgccact ggtagcttg ggcattgac ttg 576 AlaSerProPro LeuThr ThrAlaThr GlySerLeu GlyIleAsp Leu SUBSTITUTE SHEET (RULE 26) aaa gagcccatt tataca caaaatgga aaactagga ctaaagtac ggg 624 Lys GluProIle TyrThr GlnAsnGly LysLeuGly LeuLysTyr Gly get cctttgcat gtaaca gacgaccta aacactttg accgtagca act 672 AIa ProLeuHis ValThr AspAspLeu AsnThrLeu ThrValAla Thr ggt ccaggtgtg actatt aataatact tccttgcaa actaaagtt act 720 Gly ProGlyVal ThrIle AsnAsnThr SerLeuGln ThrLysVal Thr gga gccttgggt tttgat tcacaaggc aatatgcaa cttaatgta gca 768 Gly AlaLeuGly PheAsp SerGlnGly AsnMetGln LeuAsnVal Ala gga ggactaagg attgat tctcaaaac agacgcctt atacttgat gtt 816 Gly GlyLeuArg IleAsp SerGlnAsn ArgArgLeu IleLeuAsp Val .agt tatccgttt gatget caa~aaccaa ctaaatcta agactagga cag 864 Ser TyrProPhe AspAla GlnAsnGln LeuAsnLeu ArgLeuGly Gln ggc cctcttttt ataaac tcagcccac aacttggat attaactac aac 912 Gly ProLeuPhe IleAsn SerAlaHis AsnLeuAsp IleAsnTyr Asn aaa ggcctttac ttgttt aca~,~gct tca aacaattcc aaaaagctt gag 960 Lys Gl.yLeuTyr LeuPhe ThrAlaSer AsnAsnSer LysLysLeu Glu gtt aacctaagc actgcc aaggggttg atgtttgac getacagcc ata 1008 Val AsnLeuSer ThrAla LysGlyLeu MetPheAsp AlaThrAla Ile gcc attaatgca ggagat gggcttgaa tttggttca cctaatgca cca 1056 Ala IleAsnAla GlyAsp GlyLeuGlu PheGlySer ProAsnAla Pro aac acaaatccc ctcaaa acaaaaatt ggccatggc ctagaattt gat 1104 Asn ThrAsnPro LeuLys ThrLysIle GlyHisGly LeuGluPhe Asp tca aacaagget atggtt cctaaacta ggaactggc cttagtttt gac 1152 Ser AsnLysAla MetVal ProLysLeu GlyThrGly LeuSerPhe Asp agc acaggtgcc attaca gtaggaaac aaaaataat gataagcta act 1200 Ser ThrGlyAla IleThr ValGlyAsn LysAsnAsn AspLysLeu Thr ttg tggaccaca ccaget ccatctcct aactgtaga ctaaatgca gag 1248 Leu TrpThrThr ProAla ProSerPro AsnCysArg LeuAsnAla Glu aaa gatgetaaa ctcact ttggtctta acaaaatgt ggcagtcaa ata 1296 Lys AspAlaLys LeuThr LeuValLeu ThrLysCys GlySerGln Ile ctt getacagtt tcagtt ttggetgtt aaaggcagt ttggetcca ata 1344 Leu AlaThrVal SerVal LeuAlaVal LysGlySer LeuAlaPro Ile tct ggaacagtt caaagt getcatctt attataaga tttgacgaa aat 1392 SUBSTITUTE SHEET (RULE 26) SerGly ThrValGln SerAla HisLeu IleIleArg PheAspGlu Asn . 455 460 ggagtg ctactaaac aattcc ttcctg gacccagaa tattggaac ttt 1440 GlyVal LeuLeuAsn AsnSer PheLeu AspProGlu TyrTrpAsn Phe agaaat ggagatctt actgaa ggcaca gcctataca aacgetgtt gga 1488 ArgAsn GlyAspLeu ThrGlu GlyThr AlaTyrThr AsnAlaVal Gly tttatg cctaaccta tcaget tatcca aaatctcac actgccaaa agt 1536 PheMet ProAsnLeu SerAla TyrPro LysSerHis ThrAlaLys Ser aacatt gtcagtcaa gtttac ttaaac ggagacaaa actaaacct gta 1584 AsnIle ValSerGln ValTyr LeuAsn GlyAspLys ThrLysPro Val acacta accattaca ctaaac ggtaca caggaaaca ggagacaca act 1632 ThrLeu ThrIleThr LeuAsn GlyThr GlnGluThr GlyAspThr Thr 530 535. 540 ccaagt gcatactct atgtca ttttca tgggactgg tctggccac aac 1680 ProSer AlaTyrSer MetSer PheSer TrpAspTrp SerGlyHis Asn tacatt aatgaaata tttgcc acatcc tcttacact ttttcatac att 1728 TyrIle AsnGluIle PheAla ThrSer SerTyrThr PheSerTyr Ile gcccaa gaataa 1740 AlaGln Glu <210> 18 <211> 579 <212> PRT
<213> Artificial Sequence <220>
<223> Codes for a mutated Human Adenovirus type 5 fiber protein. Nucle otides corresponding to amino acids 509(G) & 510(K) of wild-type fiber were deleted.
<400> 18 Met Lys Arg Ala Arg Pro Ser Glu Asp Thr Phe Asn Pro Val Tyr Pro Tyr Asp Thr Glu Thr Gly Pro Pro Thr Val Pro Phe Leu Thr Pro Pro Phe Val Ser Pro Asn Gly Phe Gln Glu Ser Pro Pro Gly Val Leu Ser Leu Arg Leu Ser Glu Pro Leu Val Thr Ser Asn Gly Met Leu Ala Leu Lys Met Gly Asn Gly Leu Ser Leu Asp Glu Ala Gly Asn Leu Thr Ser SUBSTITUTE SHEET (RULE 26) Gln Asn Val Thr Thr Val Ser Pro Pro Leu Lys Lys Thr Lys Ser Asn Ile Asn Leu Glu Ile Ser Ala Pro Leu Thr Val Thr Ser Glu Ala Leu Thr Val Ala Ala Ala Ala Pro Leu Met Val Ala Gly Asn Thr Leu Thr Met Gln Ser Gln Ala Pro Leu Thr Val His Asp Ser Lys Leu Ser Ile Ala Thr Gln Gly Pro Leu Thr Val Ser Glu Gly Lys Leu Ala Leu Gln Thr Ser Gly Pro Leu Thr Thr Thr Asp Ser Ser. Thr Leu Thr Ile Thr _.

Ala Ser Pro Pro Leu Thr Thr Ala Thr Gly Ser Leu Gly Ile Asp Leu Lys Glu Pro Ile Tyr Thr Gln Asn Gly Lys Leu Gly Leu Lys Tyr Gly 195 ~ 200 205 Ala Pro Leu His Val Thr Asp Asp Leu Asn Thr Leu Thr Val Ala Thr Gly Pro Gly Val Thr Ile Asn Asn Thr Ser Leu Gln Thr Lys Val Thr Gly Ala Leu Gly Phe Asp Ser Gln Gly Asn Met Gln Leu Asn Val Ala Gly Gly Leu Arg Ile Asp Ser Gln Asn Arg Arg Leu Ile Leu Asp Val Ser Tyr Pro Phe Asp Ala Gln Asn Gln Leu Asn Leu Arg Leu Gly Gln Gly Pro Leu Phe Ile Asn Sex Ala His Asn Leu Asp Ile Asn Tyr Asn Lys Gly Leu Tyr Leu Phe Thr Ala Ser Asn Asn Ser Lys Lys Leu GIu Val Asn Leu Ser Thr Ala Lys Gly Leu Met Phe Asp Ala Thr Ala Ile Ala Ile Asn Ala Gly Asp Gly Leu Glu Phe Gly Ser Pro Asn Ala Pro SUBSTITUTE SHEET (RULE 26) Asn Thr Asn Pro Leu Lys Thr Lys Ile Gly His Gly Leu Glu Phe Asp Ser Asn Lys Ala Met Val Pro Lys Leu Gly Thr Gly Leu Ser Phe Asp Ser Thr Gly Ala Ile Thr Val Gly Asn Lys Asn Asn Asp Lys Leu Thr Leu Trp Thr Thr Pro Ala Pro Ser Pro Asn Cys Arg Leu Asn Ala Glu Lys Asp Ala Lys Leu Thr Leu Val Leu Thr Lys Cys Gly Ser Gln Ile Leu Ala Thr Val Ser Val Leu Ala Val Lys Gly Ser Leu Ala Pro Ile Ser Gly Thr Val Gln Ser Ala His Leu Ile Ile Arg Phe Asp Glu Asn Gly Val Leu Leu Asn Asn Ser Phe Leu Asp Pro Glu Tyr Trp Asn Phe Arg Asn Gly Asp Leu Thr Glu Gly Thr Ala Tyr Thr Asn Ala Val Gly Phe Met Pro Asn Leu Ser Ala Tyr Pro Lys Ser His Thr Ala Lys Ser Asn Ile Val Ser Gln Val Tyr Leu Asn Gly Asp Lys Thr~Lys Pro Val Thr Leu Thr Ile Thr Leu Asn Gly Thr Gln Glu Thr Gly Asp Thr Thr Pro Ser Ala Tyr Ser Met Ser Phe Ser Trp Asp Trp Ser Gly His Asn Tyr Ile Asn Glu Ile Phe Ala Thr Ser Ser Tyr Thr Phe Ser Tyr Ile Ala Gln Glu <210> 19 <211> 1740 <212> DNA
<213> Artificial Sequence SUBSTITUTE SHEET (RULE 26) <220>
<223> Codes for a mutated Human Adenovirus type 5 fiber protein. Nucle otides corresponding to amino acids 538(G) & 539(T) of wild-type fiber were deleted.
<220>
<221> CDS
<222> (1)..(1740) <223>
<400>

atg aag cgcgoa agacogtot gaagat acottcaac occgtgtat cca 48 Met Lys ArgAla ArgProSer GluAsp ThrPheAsn ProValTyr Pro tat gac aoggaa aocggtcot coaact gtgcctttt ottactcct ccc 96 Tyr Asp ThrGlu ThrGlyPro ProThr ValProPhe LeuThrPro Pro ttt gta tcoccc aatgggttt caagag agtccccct ggggtactc tot 144 Phe Val SerPro AsnGlyPhe GlnGlu SerProPro GlyValLeu Ser 35 40 45 .

ttg cgc ctatoc gaaoctcta gttaoc tcoaatggc atgcttgcg ctc 192 Leu Arg LeuSer GluProLeu ValThr SerAsnGly MetLeuAla Leu aaa atg ggcaao ggcctctot ctggao gaggooggc aacottacc toc 240 Lys Met GlyAsn GlyLeuSer LeuAsp GluAlaGly AsnLeuThr Ser caaaat gtaacc actgtgagc ccacctctc aaaaaaaco aagtca aao 288 GlnAsn ValThr ThrVa1Ser ProProLeu LysLysThr LysSer Asn g5 90 95 ataaac ctggaa atatctgca occctcaca gttacotca gaagcc cta 336 IleAsn LeuGlu IleSerAla ProLeuThr ValThrSer GluAla Leu actgtg getgcc gccgcaoct otaatggto gogggcaac acactc aco 384 ThrVal AlaAla AlaAlaPro LeuMetVal AlaGlyAsn ThrLeu Thr 115 120 1~5 atgcaa tcacag gcccogcta acogtgcac gaotocaaa cttagc att 432 MetGln SerGln AlaProLeu ThrValHis AspSerLys LeuSer Ile gocacc caagga cecctcaca gtgtcagaa ggaaagcta gocctg oaa 480 AlaThr GlnGly ProLeuThr ValSerGlu GlyLysLeu AlaLeu Gln aoatca ggCCCC CtCaCCaCC aCCgatagc agtaccctt actatc act 528 ThrSer GlyPro LeuThrThr ThrAspSer SerThrLeu ThrIle Thr gcotca ccocct ctaactact gooactggt agcttgggo attgac ttg 576 AlaSer ProPro LeuThrThr AlaThrGly SerLeuGly IleAsp Leu aaagag ccoatt tatacacaa aatggaaaa ctaggacta aagtac ggg 624 LysGlu ProIle TyrThrGln AsnGlyLys LeuGlyLeu LysTyr Gly getcct ttgcat gtaacagac gacctaaac actttgacc gtagca act 672 AlaPro LeuHis ValThrAsp AspLeuAsn ThrLeuThr ValAla Thr SUBSTITUTE SHEET (RULE 26) ggt ccaggtgtg actatt aataatact tccttgcaa actaaagtt act 720 Gly ProGlyVal ThrIle AsnAsnThr SerLeuGln ThrLysVal Thr gga gccttgggt tttgat tcacaaggc aatatgcaa cttaatgta gca 768 Gly AlaLeuGly PheAsp SerGlnGly AsnMetGln LeuAsnVal Ala gga ggactaagg attgat tctcaaaac agacgcctt atacttgat gtt 816 Gly GlyLeuArg IleAsp SerGlnAsn ArgArgLeu IleLeuAsp Val agt tatccgttt gatget caaaaccaa etaaatcta agactagga cag 864 Ser TyrProPhe AspAla GlnAsnGln LeuAsnLeu ArgLeuGly Gln ggc cctcttttt ataaac tcagcccac aacttggat attaactac aac 912 Gly ProLeuPhe IleAsn SerAlaHis AsnLeuAsp IleAsnTyr Asn aaa ggcctttac ttgttt acagettca aacaattec aaaaagctt gag .. 960 .

Lys GlyLeuTyr LeuPhe ThrAlaSer AsnAsnSer LysLysLeu Glu gtt aacctaagc actgcc aaggggttg atgtttgac getacagcc ata 1008 Val AsnLeuSer ThrAla LysGlyLeu MetPheAsp AlaThrAla Ile gcc attaatgca ggagat gggcttgaa tttggttca cctaatgca cca 1056 Ala IleAsnAla GlyAsp GlyLeuGlu PheGlySer ProAsnAla Pro aac acaaatccc ctcaaa acaaaaatt ggccatggc ctagaattt gat 1104 .

Asn ThrAsnPro LeuLys ThrLysIle GlyHisGly LeuGluPhe Asp tca aacaagget atggtt cetaaacta ggaactggc cttagtttt gac 1152 Ser AsnLysAla MetVal ProLysLeu GlyThrGly LeuSerPhe Asp agc acaggtgcc att.acagtaggaaac aaaaataat gataagcta act _1200 ~ ' , Ser ThrGlyAla IleThr ValGlyAsn LysAsnAsn AspLysLeu Thr 3g5 390 395 400 ttg tggaccaca ccaget ccatctcct aactgtaga ctaaatgca gag 1248 Leu TrpThrThr ProAla ProSerPro AsnCysArg LeuAsnAla Glu aaa gatgetaaa ctcact ttggtctta acaaaatgt ggcagteaa ata 1296 Lys AspAlaLys LeuThr LeuValLeu ThrLysCys GlySerGln Ile ctt getacagtt tcagtt ttggetgtt aaaggcagt ttggetcca ata 1344 Leu AlaThrVal SerVal LeuAlaVal LysGlySer LeuAlaPro Ile tct ggaaeagtt caaagt getcatctt attataaga tttgacgaa aat 1392 Ser GlyThrVal GlnSer AlaHisLeu IleIleArg PheAspGlu Asn gga gtgctacta aacaat tccttcctg gacccagaa tattggaac ttt 1440 Gly ValLeuLeu AsnAsn SerPheLeu AspProGlu TyrTrpAsn Phe aga aatggagat ettact gaaggcaca gectataca aacgetgtt gga 1488 Arg AsnGlyAsp LeuThr GluGlyThr AlaTyrThr AsnAlaVal Gly SUBSTITUTE SHEET (RULE 26) tttatg cctaac ctatcaget tatcca aaatctcac ggtaaaact gcc 1536 PheMet ProAsn LeuSerAla TyrPro LysSerHis GlyLysThr Ala aaaagt aacatt gtcagtcaa gtttac ttaaacgga gacaaaact aaa 1584 LysSer AsnIle ValSerGln ValTyr LeuAsnGly AspLysThr Lys cctgta acacta accattaca ctaaac caggaaaca ggagacaca act 1632 ProVal ThrLeu ThrIleThr LeuAsn GlnGluThr GlyAspThr Thr ccaagt gcatac tctatgtca ttttca tgggactgg tctggccac aac 1680 ProSer AlaTyr SerMetSer PheSer TrpAspTrp SerGlyHis Asn tacatt aatgaa atatttgcc acatcc tcttacact ttttcatac att 1728 TyrIle AsnGlu IlePheAla ThrSer SerTyrThr PheSerTyr Ile gcc caa gaa taa 1740 Ala Gln Glu <210> 20 <211> 579 <212> PRT
<213> Artificial Sequence <220>
<223> Codes for a mutated Human Adenovirus type 5 fiber protein. Nucle otides corresponding to amino acids 538(G) & 539(T) of wild-type fiber were deleted.
<400> 20 Met Lys Arg Ala Arg Pro Ser Glu Asp Thr Phe Asn Pro Val Tyr Pro Tyr Asp Thr Glu Thr Gly Pro Pro Thr Val Pro Phe Leu Thr Pro Pro Phe Val Ser Pro Asn Gly Phe Gln Glu Ser Pro Pro Gly Val Leu Ser Leu Arg Leu Ser Glu Pro Leu Val Thr Ser Asn Gly Met Leu Ala Leu Lys Met Gly Asn Gly Leu Ser Leu Asp Glu Ala Gly Asn Leu Thr Ser Gln Asn Val Thr Thr Val Ser Pro Pro Leu Lys Lys Thr Lys Ser Asn Ile Asn Leu Glu Ile Ser Ala Pro Leu Thr Val Thr Ser Glu Ala Leu SUBSTITUTE SHEET (RULE 26) Thr Val Ala Ala Ala Ala Pro Leu Met Val Ala Gly Asn Thr Leu Thr Met Gln Ser Gln Ala Pro Leu Thr Val His Asp Ser Lys Leu Ser Ile Ala Thr Gln Gly Pro Leu Thr Val Ser Glu Gly Lys Leu Ala Leu Gln Thr Ser Gly Pro Leu Thr Thr Thr Asp Ser Ser Thr Leu Thr Ile Thr Ala Ser Pro Pro Leu Thr Thr Ala Thr Gly Ser Leu Gly Ile Asp Leu Lys Glu Pro Ile Tyr Thr Gln Asn Gly Lys Leu Gly Leu Lys Tyr Gly 195 2,00 205 ., Ala Pro Leu His Val Thr Asp Asp Leu Asn Thr Leu Thr Val Ala Thr Ghy Pro Gly Val Thr Ile Asn Asn Thr Ser Leu Gln Thr Lys Val Thr Gly Ala Leu Gly Phe Asp Ser Gln Gly Asn Met Gln Leu Asn Val Ala Gly Gly Leu Arg Ile Asp Ser Gln Asn Arg Arg Leu Ile Leu Asp Val Ser Tyr Pro Phe Asp Ala Gln Asn Gln Leu Asn Leu Arg Leu Gly Gln 275 280 285 _.
Gly Pro Leu Phe Ile Asn Ser Ala His Asn Leu Asp Ile Asn Tyr Asn Lys Gly Leu Tyr Leu Phe Thr Ala Ser Asn Asn Ser Lys Lys Leu Glu Val Asn Leu Ser Thr Ala Lys Gly Leu Met Phe Asp Ala Thr Ala Ile Ala Ile Asn Ala Gly Asp Gly Leu Glu Phe Gly Ser Pro Asn Ala Pro Asn Thr Asn Pro Leu Lys Thr Lys Ile Gly His Gly Leu Glu Phe Asp Ser Asn Lys Ala Met Val Pro Lys Leu Gly Thr Gly Leu Ser Phe Asp SUBSTITUTE SHEET (RULE 26) Ser Thr Gly Ala Ile Thr Val Gly Asn Lys Asn Asn Asp Lys Leu Thr Leu Trp Thr Thr Pro Ala Pro Ser Pro Asn Cys Arg Leu Asn Ala Glu Lys Asp Ala Lys Leu Thr Leu Val Leu Thr Lys Cys Gly Ser Gln Ile Leu Ala Thr Val Ser Val Leu Ala Val Lys Gly Ser Leu Ala Pro Ile Ser Gly Thr Val Gln Ser Ala His Leu Ile Ile Arg Phe Asp Glu Asn Gly Val Leu Leu Asn Asn Ser Phe Leu Asp Pro Glu Tyr Trp Asn Phe Arg Asn Gly Asp Leu Thr Glu Gly Thr Ala Tyr Thr Asn Ala Val Gly Phe Met Pro Asn Leu Ser Ala Tyr Pro Lys Ser His Gly Lys Thr Ala Lys Ser Asn Ile Val Ser Gln Val Tyr Leu Asn Gly Asp Lys Thr Lys Pro Val Thr Leu Thr Ile Thr Leu Asn Gln Glu Thr Gly Asp Thr Thr Pro Ser Ala Tyr Ser Met Ser Phe Ser Trp Asp Trp Ser Gly His Asn Tyr Ile Asn Glu Ile Phe Ala Thr Ser Ser Tyr Thr Phe Ser Tyr Ile Ala Gln Glu <210> 21 <211> 38 <212> DNA
<213> Artificial Sequence <220>
<223> PCR primer <220>
<221> primer_bind <222> (1)..(38) <223>

SUBSTITUTE SHEET (RULE 26) <400> 21 accacaccag ctccagaggc taactgtaga ctaaatgc 38 <210> 22 <211> 38 <212> DNA
<213> Artificial Sequence <220>
<223> PCR primer <220>
<221> primer bind <222> (1) . . (38) <223>
<400> 22 gcatttagtc tacagttagc ctctggagct ggtgtgtt 38 <210> 23 <211> 38 <212> DNA
<213> Artificial Sequence <220>
<223> PCR primer <220>
<221> primer bind <222> (1) . . (38) <223>
<400> 23 acagtttcag ttttggccgg cagtttggct ccaatatc 38 <210> 24 <211> 38 <212> DNA
<213> Artificial Sequence <220>
<223> PCR primer <220>
<221> primer bind <222> (1)..(38) <223>
<400> 24 gatattggag ccaaactgcc ggccaaaact gaaactgt 3.8 <210> 25 <211> 36 <212> DNA
<213> Artificial Sequence <220>
<223> PCR primer <220>
SUBSTITUTE SHEET (RULE 26) <221> primer bind <222> (1)..(36) <223>
<400> 25 acagtttcag ttttggctaa aggcagtttg gctcca 36 <210> 26 <211> 36 <212> DNA
<213> Artificial Sequence <220>
<223> PCR primer <220>
<221> primer_bind <222> (1)..(36) <223>
<400> 26 tggagccaaa ctgcctttag ccaaaactga aactgt 36 <210> 27 <211> 36 <212> DNA
<213> Artificial Sequence <220>
<223> PCR primer <220>
<221> primer_bind <222> (1)..(36) <223>
<400> 27 gtttcagttt tggctgttgg cagtttggct ccaata 36 <210> 28 <211> 36 <212> DNA
<213> Artificial Sequence <220>
<223> PCR primer <220>
<221> primer bind <222> (1) . . (36) <223>
<400> 28 tattggagcc aaactgccaa cagccaaaac tgaaac 36 <210> 29 <211> 36 <212> DNA
<213> Artificial Sequence SUBSTITUTE SHEET (RULE 26) <220>
<223> PCR primer <220>
<221> primer bind <222> (1)..(36) <223>
<400> 29 gtttcagttt tggctgctgc aggcagtttg gctcca 36 <210> 30 <211> 36 <212> DNA
<213> Artificial Sequence <220>
<223> PCR primer <220>
<221> primer bind <222> (1) . . (36) <223>
<400> 30 tggagccaaa ctgcctgcag cagccaaaac tgaaac 36 <210> 31 <211> 36 <212> DNA
<213> Artificial Sequence <220>
<223> PCR primer <220>
<221> primer bind <222> (1) . . (36) <223>
<400> 31 gctcatctta ttatagaatt cgacgaaaat ggagtg 36 <210> 32 <211> 36 <212> DNA
<213> Artificial Sequence <220>
<223> PCR primer <220>
<221> primer_bind <222> (1) . . (36) <223>
<400> 32 cactccattt tcgtcgaatt ctataataag atgagc 36 SUBSTITUTE SHEET (RULE 26) <210> 33 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> PCR primer <220>
<221> primer bind <222> (1)..(39) <223>
<400> 33 gcttatccaa aatctcacac tgccaaaagt aacattgtc 39 <210> 34 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> PCR primer <220>
<221> primer bind <222> (1),.(39) <223>
<400> 34 gacaatgtta cttttggcag tgtgagattt tggataagc 39 <210> 35 <211> 35 <212> DNA
<213> Artificial Sequence <220>
<223> PCR primer <220>~
<221> primer bind <222> (1)..(35) <223>
<400> 35 ctaaccatta cactaaacca ggaaacagga gacac 35 <210> 36 <211> 35 <212> DNA
<213> Artificial Sequence <220>
<223> PCR primer <220>
<221> primer_bind <222> (1) . . (35) <223>

SUBSTITUTE SHEET (RULE 26) <400> 36 gtgtctcctg tttcctggtt tagtgtaatg gttag 35 <210> 37 <211> 33 <212> DNA
<213> Artificial Sequence <220>
<223> PCR primer <220>
<221> primer bind <222> (1) . . (33 ) <223>
<400> 37 ataagatttg acgaaactgg agtgctacta aac 33 <210> 38 <211> 33 <212> DNA
<213> Artificial Sequence <~220>
<2:? 3 > PCR primer <220>
<221> primer bind <222> (1)..(33) <223>
<400> 38 gtttagtagc actccagttt cgtcaaatct tat 33 <210> 39 <211> 33 <212> DNA
<213> Artificial Sequence <220>
<223> PCR primer <220>
<221> primer bind <222> (1)..(33) <223>
<400> 39 tttgacgaaa atggacacct actaaacaat tcc 33 <210> 40 <211> 33 <212> DNA
<213> Artificial Sequence <220>
<223> PCR primer SUBSTITUTE SHEET (RULE 26) <220>
<221> primer_bind <222> (1) . . (33) <223>
<400> 40 ggaattgttt agtaggtgtc cagtttcgtc aaa 33 <210> 41 <211> 33 <212> DNA
<213> Artificial Sequence <220>
<223> PCR primer <220>
<221> primer_bind <222> (1) . . (33) <223>
<400> 41 aacctatcag cttatgcaaa atctcacggt aaa 33 <210> 42 <211> 32 <212> DNA
<213> Artificial Sequence <220>
<223> PCR primer <220>
<221> primer_bind <222> (1) . . (32) <223>
<400> 42 tttaccgtga gattttgcat aagctgatag gt 32 <210> 43 <211> 10 <212> PRT
<213> Artificial Sequence <220>
<223> cRGD consensus sequence <400> 43 His Cys Asp Cys Arg Gly Asp Cys Phe Cys SUBSTITUTE SHEET (RULE 26)

Claims (62)

CLAIMS:

1. A mutated adenoviral fiber protein wherein at least one amino acid in the CD loop of a wild-type fiber protein of an adenovirus from subgroup C, D, or E, or the long wild-type fiber of an adenovirus from subgroup F, has been mutated to reduce or substantially eliminate the ability of said fiber protein to bind to the coxsackievirus-adenovirus receptor (CAR).

2. The mutated adenoviral fiber protein of claim 1, wherein said mutation substantially eliminates the ability of said protein to bind to said CAR.

3. The mutated adenoviral fiber protein of claim 2, wherein said fiber protein is an adenovirus serotype 5 fiber protein.

4. The mutated adenoviral fiber protein of claim 3, wherein said fiber protein contains at least one mutation at amino acid positions 441 and 442 of the wild-type fiber protein.

5. The mutated adenoviral fiber protein of claim 4, wherein said fiber protein further comprises a mutation at one or more of the following amino acid positions of the wild-type fiber protein: 408, 409, 460, 509, 510, 538, and 539.

6. The mutated adenoviral fiber protein of claim 4, wherein said fiber protein further comprises at least one mutation at amino acid positions 408 and 409 of the wild-type fiber protein.

7. A mutated adenovirus serotype 5 fiber protein wherein said fiber protein comprises a mutation at one or more of the following amino acid positions of the wild-type fiber protein: 460, 509, 510, 538, and 539, wherein said mutation reduces or substantially eliminates the ability of said fiber protein to bind to CAR.

8. A polynucleotide encoding the protein of claim 1.

9. A polynucleotide encoding the protein of claim 3.

10. A polynucleotide encoding the protein of claim 4.

11. A polynucleotide encoding the protein of claim 5.

12. A polynucleotide encoding the protein of claim 7.

13. An adenoviral particle comprising the fiber protein of claim 1.

14. An adenoviral particle comprising the fiber protein of claim 3.

15. An adenoviral particle comprising the fiber protein of claim 4.

16. An adenoviral particle comprising the fiber protein of claim 5.

17. An adenoviral particle comprising the fiber protein of claim 7.

18. The adenoviral particle of claim 13 further comprising a targeting ligand included in a capsid protein of said particle.

19. The adenoviral particles of claim 18 wherein said capsid protein is the mutated adenoviral fiber protein.

20. The adenoviral particle of claim 19 further comprising at least one heterologous polynucleotide.

21. The adenoviral particle of any one of claims 14-17 further comprising a targeting ligand included in a capsid protein of said particle.

22. The adenoviral particle of claim 21 wherein said capsid protein is the mutated adenoviral fiber protein.

23. The adenoviral particle of claim 22 further comprising at least one heterologous polynucleotide.

24. An adenovirus packaging cell comprising the polynucleotide of claim 8.

25. A method of making the adenoviral particle of claim 13, comprising the steps of:
transferring the adenovirus genome to be packaged in said particle into the packaging cell of claim 24;
culturing said packaging cell; and recovering an adenoviral particle produced by said cell.

26. A method of making the adenoviral particle of claim 18 comprising the steps of:
transferring the adenovirus genome to be packaged in said particle into a cell having adenovirus polynucleotides that provide proteins necessary for the replication, maturation, and packaging of said genome;
culturing said cell under conditions permitting the production of said particle; and recovering an adenoviral particle produced by said cell.

27. A method of expressing a heterologous polynucleotide in a cell comprising infecting said cell with the adenoviral particle of claim 20.

28. The method of claim 27, wherein said cell is a mammalian cell.

29. The method of claim 28, wherein said mammalian cell is a primate cell.

30. The method of claim 29, wherein said primate cell is a human cell.

31. A composition comprising the adenoviral particle of claim 18 in a pharmaceutically acceptable carrier.

32. A composition comprising the adenoviral particle of claim 20 in a pharmaceutically acceptable carrier.

33. A mutated adenovirus serotype 5 fiber protein wherein said fiber protein contains mutations at amino acid positions 408 and 409 of the wild-type fiber protein.

34. The mutated fiber protein of claim 33, wherein said protein contains deletions at amino acid positions 408 and 409 of the wild-type fiber protein.

35. The mutated fiber protein of claim 33, wherein said protein contains amino acid substitutions at amino acid positions 408 and 409 of the wild-type fiber protein.

36. The mutated fiber protein of claim 35, wherein glutamic acid is substituted for serine at position 408 and alanine is substituted for proline at position 409 (SEQ m NO:
4).

37. A polynucleotide encoding the protein of claims 33-36.

38. An adenoviral particle comprising the fiber protein of claims 33-36.

39. The adenoviral particle of claim 38, further comprising a targeting ligand included in a capsid protein of said particle.

40. The adenoviral particle of claim 39, further comprising at least one heterologous polynucleotide.

41. The adenoviral particle of claim 38, wherein at least one of the penton proteins of said particle has been modified to delete the RGD sequence.

42. An adenovirus packaging cell comprising the polynucleotide of claim 37.

43. A method of making the adenoviral particle of claim 38, comprising the steps of:
transferring the adenovirus genome to be packaged in said particle into the packaging cell of claim 42;
culturing said packaging cell; and recovering an adenoviral particle produced by said cell.

44. A method of making the adenoviral particle of claim 38, comprising the steps of:
transferring the adenovirus genome to be packaged in said particle into a cell having adenovirus polynucleotides that provide proteins necessary for the replication, maturation, and packaging of said genome;
culturing said cell under conditions permitting the production of said particle; and recovering an adenoviral particle produced by said cell.

45. A method of expressing a heterologous polynucleotide in a cell comprising infecting said cell with the adenoviral particle of claim 40.

46. The method of claim 45, wherein said cell is a mammalian cell.

47. The method of claim 45, wherein said cell is a primate cell.

48. The method of claim 45, wherein said cell is a human cell.

49. A composition comprising the adenoviral particle of claim 40 in a pharmaceutically acceptable carrier.

50. A method of enhancing adenoviral-mediated gene transfer to and expression in hepatocytes comprising the steps of administering adenoviral particles of claim 40 to said hepatocytes.

51. A method of enhancing adenoviral-mediated gene transfer to and expression in hepatocytes comprising the steps of:
preparing an adenovirus particle comprising a mutated adenovirus serotype 5 fiber protein, wherein glutamic acid is substituted for serine at amino acid position 408 and alanine is substituted for proline at amino acid position 409, and further comprising a heterologous gene; and infecting hepatocytes with said adenovirus particle.

52. A method of expressing a protein in a mammal comprising the step of administering the adenoviral particle of claim 20 or claim 40 to said mammal, wherein said particle transduces a cell in said mammal and said heterologous polynucleotide expresses said protein in said cell.

53. The method of claim 52, wherein said mammal is a primate.

54. The method of claim 53, wherein said primate is a human.

55. A method of expressing a protein in the liver of a mammal comprising administering a sufficient amount of the adenoviral particles of claim 40 for said particles to transduce cells in the liver of said mammal.

56. The method of claim 55, wherein said amount comprises approximately 1 particle per kilogram of body weight to approximately 10 13 particles par kilogram of body weight.

57. The method of claim 55, wherein said amount comprises approximately 104 particles per kilogram of body weight to approximately 10 12 particles per kilogram of body weight.

58. The method of claim 55, wherein said amount comprises approximately 108 particles per kilogram of body weight to approximately 10 11 particles per kilogram of body weight.

59. An adenoviral vector comprising the polynucleotide of any one of claims 8-12.

60. An adenoviral vector comprising the polynucleotide of claim 37.

61. The adenoviral paxticle of claims 13, 18, 20, 38, 39, 40, or 41, wherein said adenoviral particle is a replication conditional adenovirus.

62. The adenoviral paxticle of claim 61, wherein said adenovirus is an oncolytic adenovirus.