WO2005113015A1 - Enhanced gene delivery using viral vectors - Google Patents

Abstract

Lactoferrin is shown to promote adenovirus attachment and infection independently of the coxsackie and adenovirus receptor, and can be used to significantly enhance the infectivity of adenoviruses. Thus, the present invention makes available on the one hand a method for enhancing the infectivity of adenoviruses, the use of lactoferrin, and on the other hand a composition for this purpose.

Description

Enhanced gene delivery using viral vectors

Field of the invention

The present invention relates to the field of gene delivery using viral vectors, and in particular adenoviral vectors. A method for enhanced gene delivery and compositions for use in said method are disclosed.

Background of the invention

Gene therapy is an approach to treat diseases either by modifying the expression of one or more genes of an individual, or by correcting abnormal genes. By administration of DNA rather than a drug, many different diseases are currently being investigated as candidates for gene therapy. These include genetic disorders, e.g. cystic fibrosis, haemophilia, diabetes (Type 1 diabetes); metabolic disorders, e.g. diabetes (NIDDM), obesity etc. ; cardio-vascular diseases; various forms of cancer; infectious diseases such as AIDS etc. Vaccines are another important field, where methods of viral assisted gene transfer find utility. The practical use of gene therapy is however still limited due to various reasons, one of them being low gene transfer efficiency.

There are two different approaches to deliver the DNA (target genes) into the cells. The first is the usage of non-viral vectors to insert the DNA. The non-viral approach consists of methods like direct injection of the DNA, mixing the DNA with polylysine or cationic lipids that allow the DNA to be internalised. Most of these approaches have a low efficiency of delivery and transient expression of the gene. The second and more widely used approach to insert the DNA is by using viral vectors. Viruses have evolved a mechanism to insert their DNA into cells very effectively, but the side effect is that humans have evolved an effective immune response to eliminate viruses from the body.

The classical mechanism used by viruses to initiate their life cycle is mediated by a viral ligand that binds to a specific cellular receptor. Examples of such ligand-receptor interactions are HIV gp120-CD4 and influenza virus HA-sialic acid. Before such interactions take place, every virus has to pass various barriers. The innate, or non- specific immune defense in mucosal layers and body fluids constitutes an important barrier to microbial attacks in humans. Human lactoferrin (HLf) was discovered in 1959 (Groves [1]) and has been exposed to extensive studies since then. In 1966, it was demonstrated that HLf is present in various concentrations in milk (>1mg/ml), saliva (10-50 μg/ml), nasal secretions (0.1 mg/ml), respiratory mucus, tears (1.5-2 mg/ml), hepatic bile (10-40 μg/ml), pancreatic fluid (50-500 μg/ml), seminal fluid (0.5- 1 mg/ml), female cervical mucus (0.5-1 mg/ml), and urine (1 μg/ml) as reviewed by (Weinberg [2]). Blood also contains HLf but in very low concentrations (0.2 μg/ml) and it has been suggested that the plasma concentrations are the net result of the spontaneous release from the polymorphonuclear leukocytes (Baynes [3]), or activated release from these circulating cells in septic sites (Gutteberg [4]).

Since the protein is strategically situated at the mucosa, HLf plays a role in the first line of defense against microbial infections, since many pathogens tend to enter the body via the mucosa. HLf has been demonstrated to exert antimicrobial activity against numerous bacteria (Streptococcus mutans, Vibrio cholerae, Staphylococcus aureus, Listeria monocytogenes, Klebsiella pneumoniae), fungi (primarily Candida albicans), and virus (hepatitis C virus, rotavirus, poliovirus, respiratory syncytial virus, human immunodeficiency virus, and different herpes viruses (reviewed by van der Strate ef a/., [5]). The bactericidal and fungicidal activities described so far are primarily the deprivation of iron from the pathogen's microenvironment, and binding of the N-terminal region of HLf (also described as lactoferricin) to the cell walls of fungi and bacteria, which causes membrane perturbation and leakage of intracellular components (Levay [6]; Lόnnerdal [7]; Bellamy [8]). The antiviral effect of HLf is mainly related to inhibition of viral entry into host cells, either by binding to viral ligands, such as gp120 of HIV, or by binding to cellular receptors, such as heparan sulfate glycosaminoglycans, which serve as cellular receptors or co-receptors for herpesviruses and HIV, among others.

In 1953, Rowe and colleagues reported for the first time about an adenoid degenerating agent that was isolated from adenoids and tonsils in humans (Rowe [9]). Due to its tropism, this agent was later designated adenovirus. Today, 51 different human serotypes have been isolated and these are divided into 6 species (A-F) based on tropism, oncogenicity in rodents, and haemagglutination of animal red blood cells (Benkδ [10], Shenk [11], and Wadell [12]). Most human adenoviruses cause disease in the respiratory tract, intestine, eyes, urinary tract, or in lymphoid tissue (Horwitz [13]). The common adenovirus serotypes of species C (Ad1 , Ad2, Ad5, and Ad6) cause roughly 5% of all symptomatic upper respiratory tract (Brandt [14]) and 15% of lower respiratory tract (Avila [15]) infections in children younger than 5 years. Numerous early studies documented species C adenovirus isolation following explant of human tonsil and adenoid tissue to culture (Evans [16], Israel 1962, Rowe [9], van der Veen [17]). Ad2 and Ad5 have both been demonstrated to use the cellular receptor CAR

(coxsackie-adenovirus receptor) for attachment to non-polarized epithelial cell lines in vitro (Bergelson [18]; Tomko [19]) but it is unclear whether CAR mediates adenovirus infections in polarized epithelial or hematopoietic cells in vivo. Selected members of all species except species B have been demonstrated to interact with CAR on CAR- cDNA transfected cells, or with soluble CAR in slot blot assays. CAR has been demonstrated to co-immunoprecipitate with both ZO-1 and β-catenin, which are components of the tight and adherent junctions, respectively, and is localized below ZO-1 but above β-catenin in cell-cell junctional complexes (Walters [21]).

CAR is also able to form lateral intercellular homodimers in polarized airway epithelium (van Raaij [22], Cohen [23, 24], Pickles [25], Walters [21]), and is consequently an important regulator of cell-to-cell adhesion. Moreover, CAR is exposed basolaterally, but not apically in polarized airway epithelium and it is unclear whether junctional CAR is exposed to lumenal viruses (Ashbourne Excoffon [26], Cohen [23,24]). Thus, the low level of accessible CAR correlates with the poor transduction capacity of Ad5-based viral gene therapy vectors (Hutchin [27], Pickles [28]). In agreement with these findings, other receptors have been reported to facilitate species C adenovirus attachment and entry in absence of CAR in vitro (Hong [29], Dechecchi [30], Chu [31], and Huang [32]), but none of these correlates with the specific tropism exhibited by species C adenoviruses. When illuminating CAR and other molecules as species C adenovirus receptors one must also take into consideration that the cells serving as the main reservoir for persistent infections of species C adenoviruses (T-cells; Gamett [33]), express no or very little CAR (Chen [34], and Conron [35]). Recently a novel, and probably also the most correct function of in vivo fiber-CAR interactions was described: during Ad2 infections of polarized airway epithelium soluble fibers that are produced in excess in the infected cells were found to be secreted basolateraly and resolve intracellular CAR homodimers, resulting in facilitated viral escape from the basolateral region of the airway epithelium to the lumen. As a result of these findings it was postulated that successful CAR-mediated attachment and entry into target cells requires "transient breaks" in the epithelium (Walters et a/., [21]). However, here the inventors present evidence demonstrating that species C adenoviruses attach and infect epithelial as well as T-cells independently of CAR. Instead, HLf is shown to efficiently promote attachment of virions to host cells by serving as a bridge between virus particle and target cell. The viral protein responsible for the interaction with HLf is the fiber protein.

Prior art WO 94/25608 discloses a complex for gene transfer including a DNA molecule specifically and non-specifically bound to a DNA-binding protein. Additionally, it can include a chimeric compound for gene transfer. The chimeric compound has a DNA- binding element and a ligand binding element. The chimeric recombinant DNA can also include a binding protein which has a first element for binding to a receptor, a second element for binding to DNA, a third element for destabilizing endosomes and a fourth element for directing the traffic in a protein containing complex in the nucleus of a cell. The complex is contemplated for the treatment of a variety of diseases.

According to WO 94/25608, adenovirus particles enhance the uptake of intact DNA, but the adenoviruses are not used as vectors, carrying the DNA. Similarly, lactoferrin is suggested as a DNA-binding protein in a method for oral gene therapy, not for enhancing adenoviral attachment.

Also Baranov, V. S. and Baranov, A. N., Gene therapy of monogenetic hereditary disease, Voprosv meditsinskoi khimii, (2000) Vol. 6, No. 3, 279-292, mention lactoferrin in the context of gene therapy, but as a carrier protein. Summary of the invention

The present inventors have surprisingly found that lactoferrin, previously known to have antimicrobial properties, promotes adenovirus attachment and infection independently of the coxsackie and adenovirus receptor, and that lactoferrin can be used to significantly enhance the infectivity of adenoviruses. Based on this finding, confirmed in experiments conducted by the inventors, the present invention makes available on the one hand a method for enhancing the infectivity of adenoviruses, and the use of lactoferrin, and on the other hand a composition for this purpose, both as defined in the attached claims, hereby incorporated by reference. The inventive method, use and composition is preferably applied to gene transfer and/or gene therapy.

Brief description of the drawings

The invention will be described in closer detail in the following description, examples, and in the attached drawings, in which

Fig. 1A shows that tear fluid enhances the infectivity of Ad5, but not Ad37, on human corneal cells. Each green/yellow dot represents one infected cell.

Fig. 1 B shows a protein gel loaded with marker (M), tear fluid (1), lactoferrin (2), lysozyme (3) and lipophilin (4). The result confirms the identity of three of the four bands in lane (1). The fourth band is tear specific lipocalin, which however was not further investigated.

Fig. 1C shows the result of a Western blot using protein gel loaded as above, and subsequently blotted with Ad5 virus, anti-Ad5 antibodies and HRP-conjugated antibodies. The result confirms that Ad5 attaches to the four most abundant proteins present in tear fluid.

Fig. 2A shows that only soluble lactoferrin (60 μg/ml) has an effect on Ad5 infection of corneal cells. Lysozyme and lipophilin had no significant effect.

Fig. 2B proves that the effect of lactoferrin applies to multiple cell types (HCE, A549 and Hep2). Ad5 was used in the presence of lactoferrin (60 μg/ml). Fig. 3A is a bar diagram showing that lactoferrin (60 μg/ml) enhances Ad5 adhesion to different non-polarized (suspended, not adherent) cell types. The enhancement is further improved when the CAR-receptor is blocked using anti-CAR antibodies.

Fig. 3B is a bar diagram showing the relative adhesion of lactoferrin to its receptor (Lfr), CAR, and to alpha-V-beta-5-integrin on A549 (= 100 %), HCE and Hep2 cells.

Fig. 4A and B show inhibition of lactoferrin-mediated Ad5 binding to A549 and HCE cells, respectively. Recombinant, soluble knob (10 μg/ml) and serum from rabbits immunized with Ad5 fiber protein was used, the results indicating that lactoferrin binds to the knob domain of the viral fiber protein. Fig. 5 illustrates lactoferrin-mediated gene delivery. A commercial Ad5 vector expressing Green Fluorescent Protein (GFP) from CMV-promotors was used. Each green/yellow dot represents one infected cell. It is seen that lactoferrin enhances? gene delivery in a dose dependent fashion.

Description of the invention

For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

"Gene therapy" is an approach to treat diseases either by modifying the expression of one or more genes of an individual, or by correcting abnormal genes. By administration of DNA rather than a drug, many different diseases are currently being investigated as candidates for gene therapy.

The present invention relates in particular to gene therapy or gene delivery using adenoviral vectors. An adenoviral vector preferably comprises a foreign gene or nucleic acid, which will typically encode, and express within a host cell, a product that has therapeutic and/or prophylactic utility.

The term "foreign nucleic acid" is used herein to refer to any sequence of DNA or RNA, in particular DNA, functionally inserted into a vector according to the present invention that is foreign to the adenoviral genome. Such foreign nucleic acid may constitute a gene, a portion of a gene, or any other nucleic acid sequence, including but not limited to a sequence that encodes RNA, anti-sense RNA, a synthetic oligonucleotide, and/or a polypeptide. Foreign nucleic acids having therapeutic utility include genes that encode a missing or impaired gene function, and genes influencing the behavior of the cell, such as so called suicidal genes. Foreign nucleic acids having prophylactic utility include genes that encode a gene product that has an ability to prevent disease directly or indirectly, e.g. by providing a source of a polypeptide or other antigen to elicit an immune response thereto.

The term "therapeutic and/or prophylactic agent" and the term "Product having therapeutic and/or prophylactic utility" are used as equivalents and are meant to comprise inter alia antigens and immunostimulating agents, such as cytokines etc.

The terms "chimera" and "chimeric virus" means a synthetic virus, created by combining components of different viruses, natural or synthetic. The technique to produce chimeric viruses is well known to a person skilled in the art, and the task simplified by the tendency of viruses to self-assemble once the necessary viral components are present. Chimeric viruses are widely used in research applications, and e.g. in the development of vaccines, where attenuated, harmless viruses are wild type viruses in order to create new vaccines. The construction of chimeric viruses is also described in the art, see e.g. Oliveira, B.C., et a/., Construction of yellow fever- influenza A chimeric virus particles, J Virol Methods. 2002 Dec; 106(2): 185-96.

The present invention makes available a method for gene delivery or gene therapy where a virus carrying a therapeutic gene or fragment thereof is brought in contact with mammalian cells and incubated at conditions favorable to viral infection and internalization of said therapeutic gene or fragment into said cells, wherein lactoferrin is added in an amount sufficient to enhance the attachment of the virus to said cells.

Preferably, said virus is a human adenovirus belonging to species C adenovirus, and more preferably an adenovirus chosen among serotype 1 , 2, 5, and 6. According to another embodiment, the virus is a chimeric virus exhibiting the functionality of any one of adenovirus serotype 1, 2, 5, and 6 with respect to lactoferrin-mediated attachment.

According to the invention, lactoferrin is added to a concentration of at least 0.0001 mg/ml, preferably at least 0.01 mg/ml. It is contemplated that lactoferrin is added to a concentration in the interval of 0.00001 - 100 mg/ml, preferably about 0.001 to about 10 mg/ml. The concentration of lactoferrin can be determined by a skilled person, and an optimal concentration established using routine experimentation only. For each application (cell type, tissue, etc) an optimal concentration will be found between the lowest concentration having a detectable positive effect on the attachment, and the concentration over which level no further improvement can be detected, or a concentration that is toxic to the cells. It is further preferred that lactoferrin is added to said cells in a mixture with said virus particles.

The present invention also concerns the use of lactoferrin for the manufacture of a composition for the delivery of a therapeutic gene or fragment thereof, said gene or fragment contained in a viral vector. Preferably lactoferrin is used in a concentration in the interval of 0.00001 - 100 mg/ml in the composition comprising said viral vector. As defined above with respect to the inventive method, said virus or viral vector preferably is a human adenovirus belonging to species C adenovirus, and more preferably an adenovirus chosen among serotype 1 , 2, 5, and 6. According to another embodiment, the virus is a chimeric virus exhibiting the functionality of any one of adenovirus serotype 1 , 2, 5, and 6 with respect to lactoferrin-mediated attachment.

The invention also makes available compositions for use in gene transfer or gene therapy, wherein said composition comprises a virus or viral vector carrying a therapeutic gene or fragment thereof in a mixture with lactoferrin in a concentration in the interval of 0.00001 - 100 mg/ml. As above, the concentration of lactoferrin can be determined by a skilled person, and an optimal concentration established using routine experimentation only. For each application (cell type, tissue, etc) an optimal concentration will be found between the lowest concentration having a detectable positive effect on the attachment, and the concentration over which level no further improvement can be detected, or a concentration that is toxic to the cells. In addition to the vector and lactoferrin, the composition may comprise conventional solvents, preferably water, and additives, buffers, salts, etc. as well known to a skilled person.

In a composition according to the invention, said virus or viral vector is a human adenovirus belonging to species C adenovirus, more preferably an adenovirus chosen among serotype 1 , 2, 5, and 6. According to another embodiment, the virus is a chimeric virus exhibiting the functionality of any one of adenovirus serotype 1, 2, 5, and 6 with respect to lactoferrin-mediated attachment. A significant advantage of the invention is that by enhancing the infectivity, the amount of virus can be reduced. This in turn reduces the challenge to the immune system of the patient in the case of in vivo gene therapy. This is extremely important, as the immune response to viral vectors, together with insufficient infectivity, constitute the main problems in this field. In in vitro applications, the number of virus particles or their concentration can be reduced. Alternatively, the incubation time can be shortened.

Another advantage is that lactoferrin is an apparently harmless substance, which occurs naturally in the body and in body fluids in concentrations in the same range and even higher than those contemplated for use in gene delivery. It is predicted that lactoferrin, in the concentrations disclosed herein, lacks unwanted side effects.

The inventive method and composition can easily be used in existing protocols for gene delivery and/or gene therapy without any significant modifications. This is a considerable advantage, and it is predicted that the relevant authorities will readily approve new gene delivery protocols including the use of lactoferrin for both in vitro and in vivo use.

It is contemplated that further steps, such as pre-treatment, incubation etc can be used, or new steps developed without circumventing the fact that the presence of lactoferrin significantly enhances virus binding and improves the result in viral gene transfer applications.

Similarly, a person skilled in the art can optimize the concentrations or add conventional adjuvants to the lactoferrin solution, without departing from the scope of the invention.

Examples

Materials and Methods

Cells and viruses:

Human corneal epithelial (HCE) cells were grown as monolayers in supplemented hormone epithelial medium (SHEM), containing 45% Dulbecco's modified Eagle's medium (DMEM; Sigma Chemical Co., St Louis, Mo), with NaHC03 (0.75 g/liter), 45%) HAMs F-12 nutrient mixture (GibcoBRL/Life Technologies Inc., Rockville, MD) 10% fetal calf serum (FCS; Sigma), 5 ng/ml insulin (from bovine pancreas; Sigma), 0.1 μg/ml choleratoxin (Sigma), 0.2% penicillin-streptomycin (Pest; GibcoBRL), 2% Hepes pH 7.45 (Sigma), 10 ng/ml human epidermal growth factor (HEGF; Sigma), 0.5% dimethylsulfoxide (DMSO; Fluka Chemical Corp., Milwaukee, Wl), and 40 μg/ml gentamicin (Sigma). A549 cells, a cell line established from a human oat cell carcinoma of the lung, and Hep-2 cells, from epidermoid carcinoma of the larynx, were grown in DMEM, containing NaHC03 (0.75 g/liter), 10% FCS, 2% Hepes and 0.2% Pest in 37°C.

Ad37 (strain 1477) and Ad5 (strain Ad75) were propagated in A549 cells as follows: subconfluent A549 cells were inoculated at an approximate multiplicities of infection (MOI) of 100, and incubated in DMEM containing 1% FCS, for 1.5 hr in 37°C. Non- internalized virions were washed away. Cells were harvested 72 hours later, with a rubber policeman, whenever needed, and dissolved in 20 mM Tris-HCI, pH 7.5. Virions were released by repeated freeze-thaw-cycles and centrifuged to remove cell debris. The supernatant was then loaded onto a CsCI-gradient and centrifuged for 2 hr and 30 minutes, at 25000 rpm (Beckman L8-70M Ultracentrifuge). Finally, virions were dialyzed with PBS, using a NAP-5 column (Amersham Pharmacia Biotech, Buckinghamshire, England), and stored with 12.5% glycerol in -80°C.

Virus Overlay Protein Blot Assay (VOPBA): Tear fluid was induced with freshly minced unions and collected. Tear fluid proteins, as well as human lactoferrin (hLf) (Sigma), lysozyme (Sigma) and lipophilin (kindly provided by Robert Lehrer), were separated on a 4-20% gradient Criterion precast gel (BioRad, California, USA), and transferred to a PVDF membrane (Hybride-P, Amersham Pharmacia Biotech) using a Trans-Blot SD Semi-Dry Transfer cell (BioRad). The membrane was blocked with PBS in 5% non-fat dry milk (Semper, Stockholm, Sweden) overnight in 4°C. After washing 3x5 minutes in PBS-Tween (0.05%o), the membrane were incubated with 9x10⁹ Ad5 particles, diluted in 10 ml PBS-Tween (0.05%) 1% milk, for 1 hr, rocking in RT. Mouse anti-Ad1 serum (diluted 1 :5000) (ViroGen, Watertown, USA) in PBS-Tween (0.05%) and 1% milk were added to the membrane, and incubated and washed as before. Finally, horseradish peroxidase (HRP)-conjugated rabbit-anti mouse anti-antibodies (DAKO, Glostrup, Denmark), diluted 1 :5000 in PBS-Tween (0.05%) 1% milk, were added, incubated and washed as before. ECL Western blotting detection reagents were used for visualization on film (Amersham Pharmacia Biotech, Uppsala, Sweden).

Fluorescent Focus Assay (FFA):

Tear fluid, human lactoferrin (hLf), bovine lactoferrin (bLf) (Sigma), human transferrin (hTf) (Sigma), lysozyme or lipophilin, were mixed with virions (Ad5: 1.8χ10⁸ particles, Ad37: 1.4x10⁹ particles) together with 500 μl SHEM (1% FCS) on ice for 1hr. The mixtures were added to subconfluent HCE-cells in 24 well plates. Virus were allowed to bind to cells on ice, during constant agitation for 1 hr, washed twice with SHEM 1 % FCS, and incubated in 37°C for 44 hr. When experiments were done on A549 or Hep-2 cells, DMEM (1% FCS) was used instead of SHEM in all steps, and less virions were needed (A549; Ad5: 4.2x10⁶, Hep-2; Ad5: 2.2x10⁶ particles). The cells were fixed with methanol (400 μl/well), and stained first with rabbit- αAd serum, diluted 1 :200 in PBS-Tween (0.05%; Medicago AB, Uppsala, Sweden), and then with fluorescein isothiocynate (FITC) -conjugated swine-anti rabbit antibodies (DAKO, Glostrup, Denmark), diluted 1 :200 in PBS-Tween (0.05%). All antibody incubations were performed in a final volume of 400 μl for 1 hr in RT, and all washes were done in PBS-Tween (0.05%) for 2x15 min. Photos were taken with a fluorescence microscope at 20 x magnification (Axiovert 25 CFL, CarlZeiss, New York, USA). In the gene delivery experiment, AdδGFP vector (10⁴ pt /cell) was preincubated with hLf in 4°C, rocking for 1h, the same protocol was followed as in the other FFA- experiments, but instead of methanol, paraformaldehyd (2%) was used for fixation, and off course no staining with antibodies was needed.

Flow cytometry (FACS):

2x10⁵ A549 /HCE/ Hep-2 cells are resuspended in 100μl DMEM, 0.01% NaN₃, 1%BSA, +/-4μg hLf, in a 96-well plate, and incubated on ice, rocking for 1h. After centrifugation (5min, 1500rpm, +4°C), and two washes with DMEM, 0.01% NaN₃, 1%BSA, incubation (on ice, rocking for 1 h) with three different primary antibodies occurs. These antibodies are; 1) rabbit αhLf (diluted 1 :50 in DMEM, 0.01 % NaN₃, 1 %BSA), 2) mouse IgG αhuman α_vβ5 integrin (diluted 1-:500 in DMEM, 0.01% NaN₃, 1%BSA), 3) mouse αCAR (diluted 1 :200 in DMEM, 0.01% NaN₃, 1%BSA). After washing (same procedure as previously), the cells are incubated on ice, rocking for 1h, in darkness, with FITC-conjugated secondary antibodies; 1) swine αrabbit (diluted 1 :30 in DMEM, 0.01 % NaN₃, 1%BSA), and 2) rabbit αmouse (diluted 1 :20 in DMEM, 0.01 %) NaN₃, 1%BSA). The cells are washed two times as previously, resuspended in 300μl PBS, 0.01 % NaN₃, 1%BSA, and kept on ice. 2μl propidiumiodid is added 10 min before FACS analysis. Binding assay:

2x10⁵ cells/well (96-well plates) in suspension (BB: binding buffer: DMEM, pest, hepes, 1% BSA) were incubated with or without anti-CAR monoclonal antibodies, on ice. Simultanously, 2x10^{9 35}S-labelled virions/well in suspension (BB) were incubated with or without HLf, on ice. One hour later, the virion mixtures were transferred to the cells, and incubated for another hour on ice. Thereafter, non-bound virions were removed by washing, and the cell-associated radioactiviy was measured on a scintillation counter. In another binding assay, 2x10⁵ cells/well (96-well plates) in suspension (BB) were incubated with or without rabbit anti-fiber serum on ice. Simultaneously, 2x10^{9 35}S-labelled virions/well in suspension (BB) were incubated with or without soluble, recombinant fiber knobs on ice. One hour later, the virion mixtures were transferred to the cells, and incubated for another hour on ice. Thereafter, non-bound virions were removed by washing, and the cell-associated radioactivity was measured on a scintillation counter.

Results

Tear fluid promotes Ad5 infection in corneal cells

The present inventors have previously shown that three adenovirus serotypes of species D (Ad8, Ad19, and Ad37) use sialic acid as a cellular receptor for binding to host cells (Arnberg [36-40]). Unlike other adenoviruses, the tropism of these viruses is largely restricted to the eye (Ford [41]). With this in mind, the inventors set out to investigate the effect of tear fluid on adenovirus serotypes with ocular tropism (i.e. Ad37). As a control Ad5, which causes tonsillitis and respiratory infections rather than ocular ditto, was used. 1% tear fluid solutions did not affect the infectivity of Ad37 in HCE cells, whereas the same concentration efficiently enhanced the infectivity of Ad5 in HCE cells (Fig. 1A). Virus overlay protein blotting assay (Fig. 1C) revealed that Ad5 virions interacted with the four most abundant tear fluid proteins HLf (80 kDa), lipocalin 17-23 kDa, lysozyme (14 kDa), and lipophilin (Kuizenga [42], Redl [43], Gachon [44], Qu [45], and Lehrer [46, 47]), which are visualized in Fig. 1 B. When Ad5 virions were blotted to larger amounts of tear fluid proteins, weak interactions could be detected with secreted IgA (slgA) and phosphoϋpase A2 (PLA2) also (data not shown). HLf mediates Ad5 infection of host cells

To elucidate the role of specific tear fluid proteins during Ad5 infections of HCE cells, Ad5 virions were pre-incubated with purified HLf, lysozyme, lipophilin, slgA, PLA2, and human transfeπ^' (HTf). Whereas lysozyme, lipophilin, slgA, PLA2, and HTf had no effect at the concentrations used (6 μg/ml), HLf mediated a strongly increased infection (Fig. 2A and data not shown). The effect of HLf was dose dependent, and at concentrations corresponding to approximately 1/3 of that in tear fluid (0.6 mg/ml) all cells were infected (data not shown). In addition to HCE cells, HLf promoted Ad5 infection of two other cell lines also (lung epithelial cell line A549 and the larynx epithelial cell line Hep2; Fig. 2B), indicating that the effect of HLf on Ad5 infections was not restricted to ocular cells only.

HLf promotes attachment of Ad5 to target cells independently of CAR.

HLf has been shown previously to act against viruses on the level of binding to target cells (reviewed by van der Strate et al. [5]). Thus, the present inventors hypothesized that the mechanism whereby HLf promote Ad5 infection would be on the level of binding also. Using ³⁵S-labelled Ad5 virions, the present inventors found that the basal level of Ad5 binding to A549, HCE and, Hep2 cells were increased 6.2, 13.5, and, 4.5 fold, respectively in the presence of 0.2 mg/ml HLf (Fig. 3A), demonstrating that one mechanism whereby HLf mediates Ad5 infection is by promoting viral binding to host cells. However, whereas the increase in binding was obvious, it was lower than the increase seen in the infectivity assays. Walters et al. [21] showed recently that CAR is exposed basolaterally and laterally, but not apically on polarized epithelial cells. Consequently, it was hypothesized that the level of HLf-mediated infection would be higher than HLf-mediated binding, since the cells used in the infectivity model were grown as monolayers and thereby at least partially polarized, which means that lower amounts of CAR were expressed, whereas in the binding assay all cells were in suspension and CAR were exposed. To elucidate this, the inventors investigated the effect of HLf on the binding of Ad5 virions to target cells that had been preincubated with anti-CAR antibodies. Thus, the level of binding increased from 6.2 to 25.3-fold (A549), from 13.5 to 36.4-fold (HCE) and, from 4.5 to 18.1-fold (Hep2). This increase correlated with the increase obtained in the infectivity assay, and demonstrated that the mechanism whereby HLf mediate Ad5 infection is by promoting binding to target cells. Moreover, the minor differences of HLf-mediated Ad5 binding to target cells obtained without and with CAR antibodies (11 and 9.5% input of Ad5 virions bound using A549 cells; 14.5 and 16% using HCE cells; 8.5 and 6.5% using Hep2 cells), suggested that that HLf mediated attachment of Ad5 virions to target cells is independent of CAR. The relative level of HLf- mediated Ad5 binding to the cells used in this study correlated well with the relative binding of HLf to the cells. The highest binding were obtained to HCE cells, followed by A549 and Hep2 cells (Geomean = 160%, 100% and 80% to HCE, A549, and Hep2 cells respectively; Fig. 3B), which support the suggestion that the mechanism of action is to promote viral binding to target cells.

HLf promotes adenovirus infections of species C serotypes specifically.

In order to elucidate whether HLf promote infection of multiple adenovirus serotypes, the present inventors incubated HLf (0.6 mg/ml) with Ad31 (species A), Ad7 (species B1), Ad11 (species B2), Ad1 , Ad2, Ad5, and Ad6 (all species C), Ad37 (species D), Ad4 (species E) or with Ad41 (species F) prior to infection of A549 cells. Clearly, all species C adenovirus serotypes, but none of the other serotypes, infected A549 cells more efficiently in the presence of HLf (data not shown), indicating that the mechanism of action is specific for species C adenovirus serotypes.

HLf-dependent Ad5 infection is mediated by the viral fiber protein The classical mechanism whereby adenoviruses attach to host cell receptors involves an interaction between the receptor and the knob domain of the viral fiber protein. It was recently shown that Ad2-infected cells produce and basolaterally secrete a large excess of soluble fibers, which were demonstrated to facilitate subsequent escape of basolaterally secreted virions by resolving intercellular CAR dimers. This suggested that the main function of the fiber protein could be to promote release of virions from the site of infection rather then attachment and entry, and that other capsid proteins could be responsible for receptor-interactions. To assess the role of the fiber during HLf-mediated Ad5 infection, virions and HLf were co-incubated in the presence or absence of soluble knobs or anti-fiber serum prior to infection.

Clearly, both knobs and anti-fiber serum efficiently inhibited HLf-mediated Ad5- infection of A549 and HCE cells (Fig. 5). The inventors have also found that the Ad5 knob interacts with immobilized HLf in western blot (data not shown), indicating that the capsid component responsible for HLf-mediated Ad5-infection is the knob domain of the fiber polypeptide.

Fiber knob alignment

The present inventors performed alignment studies, in order to elucidate the binding mechanism accountable for the lactoferrin mediated attachment. Preliminary results indicate that three unique amino acids in the knob domain of species C adenovirus fibers may be responsible for the attachment. These amino acids were identified in serotypes 1, 2, 5 and 6. It total, adenoviruses Ad31 , 7, 11 , 1 , 2, 5, 6, 37, 4, and 41 were investigated (data not shown). Bovine lactoferrin (BLf) promotes Ad5-infection

Comparative tests using bovine lactoferrin indicate that lactoferrin of different origin may be used with equivalent results (data not shown). It is contemplated that lactoferrin of different origin can be used. It is also contemplated that species-specific lactoferrin is used, e.g. human lactoferrin in applications involving humans, bovine lactoferrin in applications involving cattle and so on. The choice of lactoferrin for a particular application can be made through routine tests, and does not require an inventive effort.

HLf promotes adenovirus-mediated gene delivery

As shown in Fig. 5, lactoferrin enhances gene delivery in a dose dependent fashion. A commercial Ad5 vector expressing Green Fluorescent Protein (GFP) from CMV- promotors was used, and the cells tested were A549 cells, cultured as described above. Each green/yellow dot represents one infected cell.

Although the invention has been described with regard to its preferred embodiments, which constitute the best mode presently known to the inventors, it should be understood that various changes and modifications as would be obvious to one having the ordinary skill in this art may be made without departing from the scope of the invention which is set forth in the claims appended hereto.

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Claims

Claims 1. A method for gene therapy where a virus carrying a therapeutic gene or fragment thereof is brought in contact with mammalian cells and incubated at conditions favorable to viral infection and internalization of said therapeutic gene or fragment into said cells, characterized in that lactoferrin is added in an amount sufficient to enhance the attachment of the virus to said cells.

2. A method for gene transfer where a virus carrying a therapeutic gene or fragment thereof is brought in contact with mammalian cells and incubated at conditions favorable to viral infection and internalization of said therapeutic gene or fragment into said cells, characterized in that lactoferrin is added in an amount sufficient to enhance the attachment of the virus to said cells.

3. The method according to claim 1 or 2, wherein said virus is a human adenovirus belonging to species C adenovirus.

4. The method according to claim 3, wherein said virus is an adenovirus chosen among serotype 1 , 2, 5, and 6. 5. The method according to claim 3, wherein said virus is chimeric virus exhibiting the functionality of any one of adenovirus serotype 1 , 2,

5, and 6 with respect to lactoferrin-mediated attachment.

6. The method according to any one of claims 1 - 5, wherein lactoferrin is added -> to a concentration between the lowest concentration having a detectable positive effect on the attachment of the virus to said cells, and the concentration over which level no further improvement can be detected, or a concentration that is toxic to said cells.

7. The method according to any one of claims 1 - 5, wherein lactoferrin is added to a concentration in the interval of 0.00001 - 100 mg/ml.

8. The method according to any one of claims 1 - 5, wherein lactoferrin is added to a concentration of at least 0.0001 mg/ml. _.

9. The method according to any one of claims 1 - 5, wherein lactoferrin is added to a concentration of at least 0.01 mg/ml.

10. The method according to claim 1 or 2, wherein lactoferrin is added to said cells in admixture with said virus particles.

11.The use of lactoferrin for the manufacture of a composition for the delivery of a therapeutic gene or fragment thereof, said gene or fragment contained in a viral vector.

12. The use according to claim 11 , wherein lactoferrin is used in a concentration between the lowest concentration having a detectable positive effect on the attachment of the virus to said cells, and the concentration over which level no further improvement can be detected, or a concentration that is toxic to said cells.

13. The use according to claim 11 , wherein lactoferrin is used in a concentration in the interval of 0.00001 - 100 mg/ml in the composition comprising said viral vector.

14. The use according to claim 11 , wherein said virus or viral vector is a human adenovirus belonging to species C adenovirus.

15. The use according to claim 11 , wherein said virus or viral vector is an adenovirus chosen among serotype 1, 2, 5, and 6.

16. The method according to claim 11, wherein said virus or viral vector is a chimeric virus exhibiting the functionality of any one of adenovirus serotype 1 , 2, 5, and 6 with respect to lactoferrin mediated attachment.

17. A composition for use in gene transfer or gene therapy, characterized in that said composition comprises a virus or viral vector carrying a therapeutic gene or fragment thereof in admixture with lactoferrin in a concentration between the lowest concentration having a detectable positive effect on the attachment of the virus to said cells, and the concentration over which level no further improvement can be detected, or a concentration that is toxic to said cells.

18. The composition according to claim 17, wherein the concentration of lactoferrin is in the interval of 0.00001 - 100 mg/ml.

19. The composition according to claim 17, wherein said virus or viral vector is a human adenovirus belonging to species C adenovirus.

20. The composition according to claim 17, wherein said virus or viral vector is an adenovirus chosen among serotype 1 , 2, 5, and 6.

1. The composition according to claim 17, wherein said virus or viral vector is a chimeric virus exhibiting the functionality of any one of adenovirus serotype 1 , 2, 5, and 6 with respect to lactoferrin mediated attachment.