This invention relates to viral vector particles, including capsid proteins with an attachment site for the specific chemical modification of the vector particles. Furthermore, the invention relates to procedures for the production of these viral vector particles. Furthermore, the invention relates to the use of these viral vector particles as a therapeutic, prophylactic or diagnostic means in humans and primates as well as other vertebrates like cattle, pigs, birds, fish, or rodents.

Description of the Invention

BRIEF SUMMARY OF THE INVENTION

The objective of this invention is to provide a procedure by which viral vector particles can specifically be chemically modified in such a way that their natural capsid- and core protein-mediated biological functions are maintained; only the receptor-binding and/or membrane fusion functions may be altered. A further objective of this invention is to provide viral vector particles that were produced with this procedure.

The present invention refers to viral vector particles, comprising capsid proteins comprising an attachment site for the specific chemical modification of said vector particles, said attachment site comprising at least one genetically introduced cysteine residue which does not naturally exist in the capsid protein. The invention further relates to viral vector particles, further comprising a coupling partner coupled to said attachment site. Another aspect of the present invention is a viral vector particle, comprising a coupling partner coupled to an attachment site, said attachment site being a cysteine residue which naturally exists in a capsid protein. The viral vector particles may comprise one or more different coupling partners. One of said coupling partner may have one or more attachment sites.

The invention further refers to viral vector particles, whereby the coupling partner(s) is/are coupled onto said attachment site via disulfide, thioester, and/or thioether bonds. The coupling partner(s) may be cell-specific ligands, polymers, especially PEG-derivatives or HPMA-derivatives, nano gold particles, fluorescence dyes, magnetic substances, or biochemically/catalytically active substances.

The invention further refers to viral vector particles, whereby the attachment site has 1, 2, 3, 4, 5, 6 or more cysteine residues and whereby two or more cysteine residues directly follow each other or are separated by 1, 2, 3, or more amino acid residues which are different from cysteine. The attachment sites preferably comprise the amino acids according to SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

Viral vector particles of the present invention derive from gene transfer vectors based on including adenovirus, adeno-associated virus, retrovirus, lentivirus, or baculovirus. In case adenovirus is used as the viral vector particle the capsid proteins are selected from Fiber protein, Hexon, Penton base and protein IX. The Fiber protein preferably comprise the amino acids according to SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15.

Another aspect of the present invention refers to a nucleic acid molecule encoding a viral vector particle according to the present invention.

Another aspect of the present invention refers to a method for the generation of a viral vector particle according to the present invention, comprising the steps of: i) generation of a viral vector particle in packaging cell lines, comprising capsid proteins comprising an attachment site for the specific modification of the vector particles, said attachment site comprising at least one cysteine residue; ii) lysing of the packaging cells and subsequent purification of said viral vector particles in buffers with a pH from 5.0 to 9.0, preferably from 6.8 to 7.4, and more preferably at 7.3, the buffer saturated with atmospheric oxygen and optionally supplemented with reducing reagents, or in oxygen-reduced or oxygen-free buffers optionally supplemented with reducing reagents or in buffers optionally supplemented with reducing reagents in an atmosphere of Ar, He, N2 or CO2; iii) contacting of coupling partners with said viral vector particles and performing a coupling reaction under formation of a thioether, disulfide, or thioester bond in oxygen-saturated buffers with a pH from 5.0 to 9.0, preferably from 6.8 to 7.4, and more preferably at 7.3, the buffer optionally supplemented with reducing reagents, or in oxygen-reduced/oxygen-free buffers optionally supplemented with reducing reagents or in buffers optionally supplemented with reducing reagents in an atmosphere of Ar, He, N2 or CO2.

A further aspect of the present invention refers to the use of the viral vector particles according to the present invention as a therapeutic, prophylactic or diagnostic means in humans, primates and other vertebrates like cattle, pigs, birds, fish, or rodents.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of this invention refers to viral vectors, comprising capsid proteins with an attachment site for the specific chemical modification of the vector particles, whereby the attachment site is a cysteine residue which does not naturally exist in the capsid protein. A further aspect refers to viral vector particles, comprising coupling partners attached to an attachement site, whereby the attachment site can be a naturally existing cysteine residue or a cysteine residue which was introduced into the capsid protein by recombinant DNA technology.

The vector particles can be produced by a combination of genetic and chemical procedures. Reactive thiol groups are introduced via cysteine residues into solvent-exposed capsid protein domains e.g. the Fiber Knob domain of Ad5 by genetic modifications. These genetic modifications permit the use of conventional production cell lines/systems and do usually not limit production yield. The genetically introduced thiol groups can optionally be kept in or transformed into a reactive i.e. reduced state by reducing reagents. The types and concentrations of the reducing reagents do not affect the integrity of the vector particles. The solvent-exposed reactive thiol groups obtained on the capsid proteins can be used for specific and covalent chemical coupling of any molecules such as ligands for tropism modification and/or polymers for shielding of the vector particles and/or marker molecules for labelling of vector particles and/or enzymes for certain catalytic activities of the vector particle surface. Either bio-irreversible covalent bonds (thioether, thioester) or bioreversible i.e. under the reducing conditions of the endosome or cytosol reversible covalent bonds (disulfides) can be formed between the coupling partner and the vector particle by choosing different chemical reagents for coupling. Furthermore, a combination of different coupling partners at different ratios and/or different chemical reagents may be used for coupling at the same time. Furthermore, coupling partners can introduce new attachment sites onto which coupling partners can be attached during further rounds of coupling.

A further aspect refers to the production of the viral vector particles according to the present invention, comprising the following steps: i) production of viral vector particle in production cell lines, comprising an attachment site for specific modification of said viral vector particle, whereby the attachment site consists of at least one cysteine residue, ii) cell lysis and/or vector particle purification in buffer solutions that are saturated with atmospheric oxygen and optionally supplemented with reducing reagents, or in oxygen-reduced/oxygen-free buffer systems that are optionally supplemented with reducing reagents, or in buffer systems saturated with Ar, He, N2 or CO2 that are optionally supplemented with reducing reagents, or combinations of the afore-mentioned buffer systems, iii) contacting the viral vector particles of step ii) with a coupling partner and performing a coupling reaction under formation of thioether, disulfide or thioester bonds in buffer solutions that are saturated with atmospheric oxygen and optionally supplemented with reducing reagents, or in oxygen-reduced/oxygen-free buffer systems that are optionally supplemented with reducing reagents, or in buffer systems saturated with Ar, He, N2 or CO2 that are optionally supplemented with reducing reagents, or combinations of the afore mentioned buffer systems with a pH in the range from 5.0 to 9.0, preferably from pH 6.8 to 7.4, and more preferably at 7.3. The isolated vector particles may be stored prior to further processing in the above listed buffers.

The basis of the described procedure is the genetic introduction of one or more reactive thiol groups into solvent-exposed domains of capsid proteins of viral vector particles via cysteine residues with suitable amino acids in their adjacency that permit control of the redox status of the cysteine residues as well as efficient alkylation/esterification. If more than one cysteine residue is being inserted, two or more cysteine residues may directly follow each other or may be separated by amino acids other than cysteines. Preferred are the sequence motives (i) LIGGGCGGGID ("1Cys") (SEQ ID NO:1), (ii) LIGCGCGCGID ("3Cys") (SEQ ID NO:2), (iii) LICCCCCID ("5Cys") (SEQ ID NO:3). The person skilled in the art can identify offhand that also motifs with differing numbers of cysteines (e.g., 2, 4, 6 or more ) and/or longer sequence motifs can be used. Furthermore it is evident that also motifs with interspersing and flanking amino acids other than glycine, serine, alanine, leucine, aspartic acid or combinations thereof may be used.

Furthermore, it is obvious that the procedure of genetic modification may also include cysteine-containing sequences with intrinsic receptor specificity or charges, e.g., sequences which are able to attach onto surface receptors of cells and/or facilitate the purification process of the vector particles.

The cysteine residues may for example be inserted into the solvent-exposed HI-loop of the Knob-domain of the capsid protein Fiber of adenovirus serotype 5. The person skilled in the art can identify offhand that alongside the solvent-exposed domains of the HI-loop in the Fiber protein also solvent-exposed areas in other regions of the Fiber protein as well as solvent-exposed regions in capsid proteins such as Hexon, Penton base and Protein IX are suitable for the insertion of the afore mentioned or similar motifs. Furthermore, it is obvious that not only adenovirus vectors may be used and in the used viruses potentially naturally existing and accessible cysteines can be modified with the invention-relevant procedure. As an example the VP1, VP2, and VP3 proteins of Adeno-associated virus (AAV) and its subtypes can be mentioned, as well as the env protein of retroviruses.

Additionally, the procedures apply to adenovirus vectors that are modified in such a manner that they no longer bear the natural Fiber protein but other trimerizing proteins for partial or complete substitution of the homotrimeric Fiber. Furthermore, the procedures apply to hybrid vectors which are composed of capsid proteins derived from different virus types, e.g. hybrid vectors composed of adenovirus and reovirus proteins. Furthermore, the procedure applies to chimeric vectors that have been composed of different Ad serotypes. Furthermore, the procedure applies to vectors which lack natural capsid components, e.g., Ad vectors that lack the fiber protein or parts of the fiber protein. In addition, the procedure applies to chimeric virus vectors other than adenovirus that are composed of different serotypes and to pseudotyped retro- and lentiviral vectors.

The cysteines may be incorporated into the vector particles by established procedures of recombinant DNA technology. For this purpose nucleic acid sequences encoding the cysteine sequence motifs are being incorporated into polynucleotide sequences which encode for the capsid protein of the vector particle to be modified. The thereby modified polynucleotide sequences are used for the production of the vector particles in production cell lines, e.g., N52E6 cells, 293 cells or PER.C6 cells, e.g., in case of Ad vector particles. The person skilled in the art is familiar with cell lines and production procedures that are used for production of adenoviral vectors and for production of other viral vectors such as for example vectors based on adeno-associated virus, retrovirus or lentivirus. In case of Ad vector particles, the nucleic acid is first of all transfected into the production cells (e.g., by the Ca-phosphate method). Subsequently, the production cell lines produce vector particles that can be harvested by cell lysis in a lysis buffer which usually is saturated with atmospheric oxygen such as phosphate-buffered saline (PBS) or in cell culture medium. Since the amounts of obtained vector particles are usually relatively low in this first step of production, the vector particles obtained by lysis are used again to infect production cell lines in order to obtain larger vector particle amounts. This is done by adding the vector particles obtained by lysis and optionally purified and/or concentrated to the production cells which are to be infected. This procedure is repeated with increasing production cell numbers up to 15 times (preferably 3-10 times). This process is called serial amplification. The buffers that are used to lyse the cells may contain reducing reagents such as tris-(2-carboxyethyl)-phosphine TCEP (1-10 mM), dithiothreitol DTT (1-10 mM), ascorbate (10-100 mM) or others. Additionally during serial amplifications right before the infection of production cells with vector particles antioxidative reagents such as ascorbate (50-100 mM) or vitamine E (1-100 mM) may be added to the cell culture medium in order to avoid the formation of vector particle aggregates prior to infection in the oxidative milieu of the cell culture medium.

Alternatively to the use of lysis buffers saturated with atmospheric oxygen optionally supplemented with reducing reagents during serial amplifications degassed buffer systems may be used, which may be obtained by vacuum, sonification, or a combination of both. Furthermore buffer systems may be used which were oxygen-depleted or oxygen-reduced by the application of argon, carbon dioxide, nitrogen or helium. Reducing reagents may also be added to the degassed buffers. Obviously all combinations of the mentioned procedures may be applied.

The buffer systems which are used for cell lysis may also be used for all purification and concentration procedures following the lysis of production cells at and after serial amplifications of the genetically capsid-modified vector particles. This also includes all purification and buffer exchange procedures prior to chemical modification of the vector particles (e.g., CsCl step gradient centrifugation, desalting by molecular sieves, dialysis). One must pay close attention to the application of materials and buffer systems which are free of divalent, metalic cations such as Mg2+ or Mn2+ in order to ensure stability and reactivity of the thiol groups to the vector particles. Chelating reagents such as EDTA or EGTA may be added to the buffers. All mentioned buffer systems are also used for vector particle storage as well as for purification and storage of the chemically modified or partially chemically modified vector particles.

By thiol-specific, chemical procedures various molecules (further on referred to as coupling partners) are attached to the vector particles by the formation of covalent chemical bonds (further on referred to as "coupling"). Here it is essential that the integrity of the vector particles as well as the natural capsid- and core protein-mediated vector particle functions are being maintained; only the natural receptor binding and/or membrane fusion functions may be altered. It is equally essential to maintain the structural integrity of the coupling partners.

The chemical modifications are carried out in buffer systems with a pH range from pH 5.0 to 9.0, preferably from pH 6.8 to 7.4, and more preferably 7.3. The chemical modifications can be carried out in a buffer solution saturated with atmospheric oxygen as well as with one or more of the previously mentioned buffer systems. The preferred buffer system is an oxygen- and metallic cation-free buffer containing 100 mM phosphate-buffered saline (PBS) solution. The person skilled in the art is familiar with the use of other inert buffer systems such as those based on HEPES. Furthermore, chemical reactions that are performed without changing the pH can also be performed in water. The used buffer system and the used reaction tube shall be free of divalent metallic cations such as Mg2+ or Mn2+ in order to ensure stability of the reduced thiol groups. Alternatively, chelators such as EDTA can be applied in order to stop undesirable interactions with thiol groups and divalent metal cations.

The coupling to genetically inserted thiols of the vector particles may take place through the formation of thioethers or thioesters (bio-irreversible covalent) or through formation of disulfide bonds (bioreversible). The formation of thioethers takes place with reagents such as maleimide derivatives, the formation of disulfide bonds either by reduced cysteines within the coupling partner or disulfide exchange reaction of cystin derivates or dithiopyridyl derivatives. The person skilled in the art is familiar with the reaction mechanisms and velocities. This knowledge permits variants of the coupling procedure according to the specific requirements of the coupling partners.

The coupling partners can be coupled directly to the vector particles by making use of intrinsic natural characteristics of the coupling partner. This can for example take place using naturally existing reduced thiol groups present in the coupling partner or by adding reducing reagents to the coupling partner to obtain reduced thiol goups e.g. from disulfide bridges present in the coupling partner. Furthermore, disulfide exchange reactions can be applied for direct coupling of the coupling partners.

Additionally, one may also apply genetically modified coupling partners such as recombinant proteins for thiol-specific coupling to vector particles, which received by genetic modification single cysteines or disulfide bridges for coupling via disulfide bonds or disulfide exchange reactions.

The coupling partners can also be coupled to the thiols on the surface of the vector particles after covalent chemical modification of the coupling partner. Here the coupling partners are chemically modified in such a way, that after this modification they posess covalently-coupled, thiol-specific reactive groups which in a subsequent step are applied for specific coupling onto the thiol groups of the vector particles. Thiol-specific reactive groups that can be inserted by chemical modification of coupling partner are for example maleimide groups for the formation of thioethers or dithiopyridyl groups for the formation of disulfide bridges or iodoacetyl derivatives for the formation of thioesters. These can be inserted by coupling reagents such as N-[E-maleimidecaproyloxy]succinimidester (EMCS) or Succinimidyl-6-[3-(2-pyridyldithio)-propionamido]hexanoat (SPDP) into the coupling partner. The person skilled in the art is familiar with the application of further reagents.

The chemical modification of the coupling partners for coupling onto the thiols on the vector particle surface can also be used to insert chemical groups that are not directly involved in the coupling process and which serve as spacers between coupling partners and vector particles. An example is the use of N-hydroxysuccinimid-polyethylenglykol-3400-maleimide (NHS-PEG3400-Mal). The use of spacers in the chemical modification of one or more coupling partners may serve to increase reaction efficiency by reduced steric hindrance as well as to increase the potency of the coupling partner by efficiently exposing its receptor-binding domains or to introduce further reactive groups for continued coupling. Obviously, apart from NHS-PEG-3400-Mal also other spacers can be used.

Clearly, one can also use coupling partners that themselves can bear specific reactive groups for the coupling of a second coupling partner in chronological order. Furthermore it is obvious that simultaneous coupling of several different coupling partners onto the vector particles can also occur.

The coupling partners for thiol-specific coupling onto the vector particles can fulfil different functions.

The first function consists in the mediation of new receptor specificities for retargeting of the vector particles which were modified in this manner. For this purpose proteins like transferrin can be applied whereby the characteristics of the coupling partners regarding receptor binding are transferred onto the vector particles by covalent coupling. It is obvious to the person skilled in the art that not only proteins with certain receptor specificities can be used here (e.g. transferrin, Epidermal Growth Factor EGF, basic Fibroblast Growth Factor bFGF), but also molecules of other substance categories. As an example mono- or polysaccharides such as galactose can be mentioned here which can mediate specific receptor binding. A further example for the application of coupling partners different from proteins are steroid hormones, which can also mediate receptor specificity. Furthermore, molecules with receptor binding characteristics that were designed with rational methods such as Molecular Modelling or derivatives of these and molecules that have been chemically synthesized or have been isolated from natural materials may be used for coupling. Also more complex molecules assembled of numerous subunits which may be covalently or even non-covalently linked e.g. viruses or virus vector particles may be used as coupling partners to be coupled to thiol groups on the vector particle surface. The vectors according to the present invention may also be coupled with coupling partners whereby developing a tropism for more than one cell type. Coupling of coupling partners to cysteine residues on the vector particle surface may also be performed before, at the same time, or after chemical modifications of the vector particle surfaceinvolving reactive groups different from thiols. For example before, at the same time, or after amino-PEGylation with amine-reactive electrophilically activated esters or before, at the same time, or after surface modification with amine-reactive HPMA thusly obtained vector particles might be subjected to thiol modification.

The second function consists in a steric shielding of the chemically modified vector particles by the coupling partners in order to avoid for example unwanted interactions with antibodies or the complement system or cellular components of the immune system. For this purpose derivatives of synthetic polymers such as polyethylene glycol (PEG) with different molecular weights and chain lengths can be applied. A further example for a synthetic polymer, whose derivatives are suitable for vector particle shielding and may thiol-specifically be coupled by one of the above described mechanisms onto the genetically modified vector particles is pHPMA (Fisher, 2001). Furthermore, molecules which confer new receptor specificities like proteins can themselves be used for shielding of vector particles.

The functions of mediating new receptor specificities by a coupling partner and shielding may be united by simultaneous or chronological coupling of two or more coupling partners onto the same vector particle, whereby tropism is created on the one hand and shielding is created on the other hand. Here it is obvious that both coupling partners can directly be coupled onto reactive thiol groups on the capsid surface or that one coupling partner offers reactive groups to the specific coupling of the second coupling partner onto the first. These reactive groups can for example be thiol, amino or carboxyl groups. Furthermore, it is obvious that a single coupling partner which combines both functions, the retargeting and the shielding, can also be used.

A third function of the coupling partners may be the labelling of the vector particles for example for analytic gene transfer with e.g. a fluorescence dye or nano gold particles as analytical markers.

A fourth function may be the possibility to alter the physical properties of the vector particles by for example coupling magnetic coupling partners to the vector particles and using the new physical properties like magnetism for, e.g., physical transduction methods or physical purification methods based on the physical properties conferred by the particular coupling partner.

A fifth function may be the possibility to alter the biochemical properties of the vector particles aside from receptor binding, i.e., to confer enzymatic activities to the vector particle by coupling enzymes or catalytically active fragments of enzymes like DNA recombinases or proteases onto the vector particles and transport these enzymatic or catalytic activities into target cells.

One prerequisite for the invention-relevant modification of vector particles is the availability of cysteine residues in solvent-accessible domains of the surface and/or the capsid proteins. Here it is irrelevant whether the viruses have an envelope or not. It is crucial that by reduction and/or alkylation/esterification of the thiol groups none of the biological vector functions are affected, only receptor-binding and/or membrane fusion features may be altered.

The invention-relevant modification of vector particles by specific coupling of ligands onto thiol groups which were genetically inserted into solvent-exposed domains, may be achieved for all viral vectors. The person skilled in the art is familiar with the existence of vectors that are based on a large number of different viruses. The viruses can be found in commonly accessible text books such as for example Fields, Virology. Especially interesting for gene therapy are currently vectors which are based on adenoviruses, adeno-associated viruses (AAV) or retroviruses and lentiviruses, and exist in different, with some viruses like AAV in many types and serotypes. For the invention-relevant modification it is required that thiol groups which are genetically inserted into solvent-exposed domains of capsid proteins and/or which are naturally available, are being kept in a reduced, reactive condition with mild reducing reagents and/or by the afore mentioned buffer systems or that they are being transferred into this condition.

The respective regions being suitable for the thiol specific coupling are positioned on the solvent-exposed domains of the viral capsid proteins so that the coupling of the coupling partners may be performed.

Example for further attachment sites for genetic insertion into certain loci of capsid proteins of the adenovirus type 5 are listed in the following table -- see Original Patent.

The modified viral vector particles according to the present invention can especially be applied for therapeutic purposes for gene therapy or vaccination or for functional and diagnostic analysis in vivo in humans, primates, or other vertebrates like cattle, pigs, birds, fish or rodents or in vitro in tissue or cell cultures comprising cells from vertebrates, in particular from humans and primates like cattle, pigs, birds, fish or rodents. Disorders can be inherited disorders which are for example caused by a mutation in one gene. Furthermore, the modified viral vector particles according to the present invention can be used for the treatment of acquired disorders such as for example tumor diseases or disorders of the central nervous system such as Parkinson syndrome. In these situations, i.e. in genetic disorders or in acquired disorders, modified viral vector particles according to the invention and carrying one or more therapeutic nucleic acids are used to enable improved and/or selective transduction of cells in vitro and in vivo, in order to allow expression of one or more therapeutic nucleic acids in these cells. The person skilled in the art is familiar with the use of viral vectors for the treatment of genetic or acquired disorders and textbooks and publications can be consulted for the selection of specific nucleic acids for the treatment of genetic or acquired disorders. The person skilled in the art is also familiar with ways to administer modified viral vector particles to individuals/animals, such as for example by systemic administration (intravenously, intraarterially), by administration through body orifices or by local needle or katheter injection into a tissue or organ. Additionally, the invention-relevant modified viral vector particles can also be applied as vaccines for example for prophylactic vaccination against HIV or other infectious diseases or as tumor vaccine. In fact, the use of modified viral particles for prophylactic or therapeutic vaccination against infections or neoplastic disorders is a preferred application. It is well known to the scientific community that the use of viral vectors for vaccination is frequently impaired or even prevented by pre-existing either B-cell or T-cell-mediated immunity that is directed to the particular vector that is used to deliver the particular gene. Also, even if in an individual there are no pre-existing antibodies to the viral vector that is used for vaccination, upon a first application of the vector usually a strong immune response is raised, preventing a second administration of the same viral vector.

The present invention enables strategies to circumvent these problems by thiol-specific coupling of either shielding reagents such as polyethylene glycol or targeting ligands, or both, to the surface of the viral vector particle, thereby preventing the recognition of viral vector surface structures by the immune system and at the same time targeting the vector to the cells and/or organs of interest.

The transgenes that may be expressed by the vector particle genome are not critical for the invention. Here it may concern for example muscle proteins, clotting factors, membrane proteins or cell cycle-regulating proteins. Example for a muscle protein is dystrophin, an example for a secreted protein is the clotting factor VIII, an example for a membrane protein is the cystic fibrosis transmembrane regulator protein (CFTR). Furthermore it may concern genes that originate from pathogens such as for example the AIDS virus HIV or animal viruses or parasites and are being expressed by the viral vector particle as a vaccine.

The use of the invention-relevant modified viral vector particles may occur in vitro or in vivo. In vitro gene transfer occurs outside of the body for example by adding vector particles to tissue or cell culture cells. In in vivo gene transfer vector particles are being applied in different ways, depending on the tissue that is to be transduced. Examples for the ways in which vector particles can be applied are injection into the arterial or venous vessel system, direct injection into the appropriate tissue (for example lung, liver, brain, muscle), instillation into the appropriate organ (for example lung or gastrointestinal tract) or direct application onto a surface (for example skin or bladder). The vector particles used for this purpose are chemically modified with, e.g., the ligand or shielding polymer by thiol-specific coupling of the ligand to the vector particle surface, in such a way that allows for ligand-specific interaction with its target receptor and subsequent particle uptake.

The basis of this procedure is the invention-relevant combination of genetic and chemical modifications of a solvent-exposed domain of a capsid protein of viral vectors whereby the chemical modification occurs through formation of covalent and optionally bioreversible bonds. A central aspect of the procedure is to maintain the integrity and the natural and capsid and core protein-mediated biological functions of the modified vector particles; only the receptor binding characteristics/membrane fusion characteristics may be altered. The basic advantages are summerized as follows: (i) vector particle production for the chemical capsid modification with conventional procedures through high yields (ii) high flexibility with the use of coupling partners, particle shielding, labelling or giving new physical or biochemical/catalytical characteristics (iii) high specificity of the chemical capsid modifications with optional bioreversibility.

Advantageous is also (1) the selective genetic insertion of reactive thiol groups onto the capsid surface before the chemical modification of the vector particles, (2) the potential use of sequences for genetic modification of the vector capsids without known receptor binding characteristics, (3) the post-productional, covalent and optionally bioreversible chemical modifications of at least one part of the genetically introduced cysteines, (4) the possibility for covalent and thus stable coupling, (5) the use of sequences for the genetic modifications as a basis for chemical modification for which no natural function is described, (6) the use of procedures that may specifically and solely use the genetically introduced reactive thiol groups without covalently modifying any wildtype existing amino acids, (7) the use of procedures that specifically modify the oxidative status of the vector particles, (8) the avoidance of crosslinking within the capsid through the high specificity of the used chemical reactions, (9) the possibility for the formation of bioreversible, i.e. in the reducing milieu of the endosome or cytosol reversible (disulfide) bonds, (10) the use of small reactive groups (e.g. maleinimides) for the chemical modification of the vector particles by the formation of covalent bonds, (11) the possibility for the use of non-peptide/non-protein coupling partners, (12) the possibility of shielding of the vector particles through the choice of appropriate coupling partners.

It is essential for the invention that with the present procedure for the modification of vector particles for gene transfer the natural capsid- and core protein-mediated biological functions of the vector particles are entirely maintained; only the receptor binding and/or membrane fusion characteristics may be altered.

The possibility of using thiol groups of cysteine residues that were genetically introduced into the vector particle for the chemical modification of the vector particles by changing their oxidative status and maintaining their integrity and function was unexpected and surprising for the following reasons: the invention-relevant procedures allow for the application of reducing reagents with subsequent alkylation of the reduced thiol groups for the specific chemical modification of the solvent-exposed cysteine residues of the genetically modified vector particles. Surprisingly the inventors of the present application could not observe alkylation of the viral cysteine protease p23 of Ad5 neither for vector particles with cysteine residues that were genetically introduced into the capsid proteins nor with genetically unmodified vector particles after chemical reduction and alkylation with monomaleimide nanogold particles. Furthermore, no change of infectivity was detected for particles that had been reduced and subsequently treated with alkylating reagents as compared to untreated control particles. According to the present standard of knowledge one would have expected that after applying reducing reagents and alkylating reagents not only the solvent-exposed thiols which were genetically inserted, but additionally also naturally existing thiols present in the capsid proteins Hexon, Penton base and Fiber as well as core protein pV would have been alkylated. Surprisingly the inventors discovered that after reduction of adenoviral vector particles without genetic modification none of these proteins was alkylated by monomaleimide nanogold particles. However, genetically introduced, solvent-exposed cysteine residues could be alkylated with the same procedure efficiently and highly specific. The selective and specific alkylation of genetically inserted cysteine residues is one of the central tasks of the procedure described here.

Claim 1 of 12 Claims

1. An infectious adenovirus viral vector particle comprising an adenovirus capsid Fiber protein, comprising the amino acid sequence set forth as SEQ ID NO: 13 and comprising an attachment site for the specific chemical modification of said vector particle, said attachment site comprising the amino acid sequence set forth as SEQ ID NO: 1.
 

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