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WO2016037297A1 - Method for producing recombinant proteins in the mammary glands of mammals by transforming the mammary gland epithelium with adeno-associated vectors - Google Patents

Method for producing recombinant proteins in the mammary glands of mammals by transforming the mammary gland epithelium with adeno-associated vectors Download PDF

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Publication number
WO2016037297A1
WO2016037297A1 PCT/CL2015/000047 CL2015000047W WO2016037297A1 WO 2016037297 A1 WO2016037297 A1 WO 2016037297A1 CL 2015000047 W CL2015000047 W CL 2015000047W WO 2016037297 A1 WO2016037297 A1 WO 2016037297A1
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Prior art keywords
aav
production
recombinant proteins
milk
interest
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PCT/CL2015/000047
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Spanish (es)
French (fr)
Inventor
Oliberto Sanchez Ramos
Jorge Toledo Alonso
Ivan GONZALEZ CHAVARRIA
Emilio SALGADO ROJAS
Daniel SCHULZ DURAN
Frank Camacho Casanova
Oscar CABEZAS AVILA
Florence HUGUES SALAZAR
Mauricio GONZALEZ OYARZUN
Alexis Salas Burgos
Raquel MONTESINO SEGUÍ
Paulina SAAVEDRA SIEYES
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Universidad De Concepcion
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Publication of WO2016037297A1 publication Critical patent/WO2016037297A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/864—Parvoviral vectors, e.g. parvovirus, densovirus

Definitions

  • bioactive proteins proteins that in addition to being long and expensive, entails the risk of transmission of infectious agents, such as the causes of AIDS and hepatitis.
  • transgenesis lies in the fact that it is possible to create a transgenic population from the founding animal through natural reproduction.
  • G0 initial 0
  • transgenic progeny does not express the exogenous protein at the same levels as the original founder.
  • Somatic cloning as an alternative route for the production of transgenic animals allows some of the above limitations to be overcome, however, the high cost as well as the inefficiency of this technique undermine its practice in a more routine way.
  • the use of transgenic mammals as biofactories also involves the construction of complex expression cassettes, where the exogenous gene is linked to regulatory sequences that only allow its expression in the mammary glandular epithelium during lactation. In many cases these regulatory sequences do not work efficiently, causing small amounts of recombinant protein to be synthesized in other tissues, with a detrimental effect on animal health.
  • EGM mammary glandular epithelial cells
  • Patent document WO 2004034780 A2 protects a gene transfer procedure to the mammary glandular epithelium that is based on the use of adenoviral vectors as a vehicle for gene transfer, at levels greater than 1 g / L.
  • the technology is efficient.
  • the expression of transgenes from adenoviral vectors is generally lost in about ten days. This is basically due to an immune response that rises against infected cells because of the immunogenicity of viral proteins expressed in said cells.
  • An additional limitation of this process is that animals cannot be reused because the immune response induced by the first viral administration totally neutralizes any subsequent transduction process.
  • Adeno-associated viruses constitute a group of vertebrate viruses, of the Parvoviridae family and of the genus Dependovirus.
  • AAV is a very simple virus between 45 and 60 nm in diameter, which contains a single stranded linear DNA genome. The virus requires co-infection with adenovirus or others to replicate.
  • AAV is widespread in the human population but is not associated with any known disease.
  • the organization of the AAV genome is extremely simple, comprising only two genes: (i) rep, which encodes a family of overlapping proteins involved in replication and integration; (ii) cap, which encodes a family of three viral structural proteins.
  • the ends of the genome are composed of repeated and inverted sequences (ITR) of about 145 nucleotides. Wild-type AAVs can integrate their DNA into the host's chromosome in the absence of the helper virus. This integration may occur more frequently in regions of chromosome 19 and is dependent on the presence of the rep protein.
  • AAV vectors contain only viral ITR viral sequences. Among these sequences the desired expression cassettes can be inserted. The genome of these vectors should not exceed the original viral genome size (4,700 kb) to Do not affect your packaging efficiency.
  • AAV it is necessary to provide trans and cap proteins in trans, which is achieved through the use of a helper virus, plasmids, or a complementation cell line (Ponnazhagan, S; 2001).
  • helper virus, plasmids, or a complementation cell line Ponnazhagan, S; 2001.
  • the main advantages of AAV-based vectors are that they have a broad tropism, are very poorly immunogenic and are able to ensure sustained expression of the transgene for long periods of time (Biolabs vector; 2007).
  • Figure 1 The figure shows a general scheme of the AAV-EPOFc vector (A) and the modeling of the designed EPO-Fc molecule (B)
  • Figure 2 The figure shows the western blot analysis of the EPO-Fc protein expressed in cell cultures transduced with the AAV-EPOFc vector using a specific monoclonal antibody against human EPO.
  • FIG 3 The figure shows an image of the mouse mammary glandular epithelium that fluoresces fluorescently by action of the control vector AAV-GFP that induces the production of the GFP report protein in the transformed epithelial cells (A); immuno-identification of the recombinant EPO-Fc protein from milk samples of mice transduced with the AAV-EPOFc vector is also shown, by a western blot (B) assay; The quantification of EPO-Fc expression levels in the milk of mice transduced with the AAV-EPOFc vector is shown by a human EPO-specific immunoassay (ELISA).
  • A immuno-identification of the recombinant EPO-Fc protein from milk samples of mice transduced with the AAV-EPOFc vector is also shown, by a western blot (B) assay
  • B western blot
  • the quantification of EPO-Fc expression levels in the milk of mice transduced with the AAV-EPOFc vector is shown by a
  • Figure 4 The figure shows the immuno-dentification of the expression of EPO-Fc produced in the milk of goats transduced with the AAV-EPOFc vector. Five goats were tested during an active lactation period (100 days) and milk samples were collected at 10 day intervals for each animal. The amount of recombinant EPO-Fc produced by each animal was quantified by ELISA (A), and the presence of EPO-Fc in milk samples was evaluated by a western blot assay.
  • the present invention corresponds to a method for the production of recombinant biomolecules; the mammary gland being used as bioreactor and AAV vectors as vehicles for gene transfer.
  • This method gives solution to the problems associated with the generation of a transgenic animal, and in addition, it allows solving the limitations of all the previous strategies of transduction of the mammary glandular epithelium.
  • the general procedure object of the present invention costs the following steps:
  • Cloning of the gene of interest in the qenoma of an AAV vector involves the construction of an expression cassette for the protein of interest flanked by the repeated and inverted sequences of an AW vector. The procedure can be performed using conventional molecular biology tools or can be obtained by chemical synthesis. The total size of the construct should not exceed 4.7 kb.
  • AAV vectors can be generated by any of the procedures available in the state of the art. Assistive virus-free systems can be used, based on transient transfection of a plasmid cocktail where one of the plasmids contains the genome that must be packaged, while the rest provide trans and cap genes in trans. Stably producing cell lines of the AAV vector, or insect cell production systems, can also be employed, using baculovirus as helper vectors.
  • This step should be repeated once or twice more to guarantee the total Rinse the mammary gland and distension of the ducts and alveoli.
  • an isoosmotic solution PBS, 0.9% NaCI, 5% glucose, HBS or tissue culture medium can be used.
  • the infusion of the solution containing the viral particles is carried out directly through the nipple canal.
  • the viral concentration in the solution to be infused can be variable although concentrations in the range between 8 and lxlO lxlO 10 pfu / ml are preferred.
  • the optimal volume of the infusion may vary depending on the size of the udder.
  • a cannula attached to a syringe or a peristaltic pump can be used.
  • the infusion should be done slowly, and during and after the infusion massages should be given to ensure that the solution is distributed homogeneously and reaches all of the mammary epithelial cells.
  • the AAV vector can infect mammary glandular epithelial cells using heparan sulfate molecules (HSPG) as the primary receptor, and as secondary receptors to integrins ⁇ ⁇ ⁇ 5 and fibroblast growth factor receptor type 1 (FGFR-1 ). To ensure access of the AAV vector to its receptors, it is advisable to supplement the infusion solution with a chelating agent that facilitates the temporary rupture of the tight junctions between the cells that make up the mammary glandular epithelium.
  • HSPG heparan sulfate molecules
  • FGFR-1 fibroblast growth factor receptor type 1
  • l, 2-bis (2-aminophenoxy) ethane-N, N, N ', N'-tetraacetic acid BAPTA
  • dimercaptosuccinic acid DMSA
  • diethylenetriamine pentaacetic acid DTPA
  • acid 2 can be used 3-dimercapto-l-propanesulfonic acid (DMPS), tri-acetic nitrile acid (NTA), ethylene glycol tetraacetic acid (EGTA), dimercaprol (INN) or, ethylene diamine tetraacetic acid (EDTA).
  • Milk collection Milk, from treated animals, can be obtained by conventional methods of manual or mechanized milking. It will be collected from the second day of the procedure until the levels of expression are attractive from an industrial point of view (usually 100-140 days).
  • Purification of the protein of interest from milk Most of the milk is stored at -20 ° C while small samples are used to detect and quantify the protein of interest with the use of techniques commonly known (eg ELISA, Western blot or biological activity). Serums containing appreciable amounts of the protein of interest are mixed and used as active raw material for purification of the heterologous protein. The purification process can vary considerably depending on the protein in question.
  • the procedure is robust, guaranteeing high levels of recombinant protein expression and ensuring sustained production for periods longer than 100 days.
  • the method of the present invention involves the infusion of a solution containing AAV vectors directly through the nipple channel.
  • Animals should preferably be ruminants (eg bovines, sheep or goats). Animals can be subjected to a hormonal induction of mammogenesis and lactation or animals that are in the natural phase of lactation can be used.
  • the transgenes are transferred into the mammary secretory cells where they are transcribed and where the translation and post-translational processes that the protein of interest requires before being secreted in the milk.
  • milking the animal eg cow, sheep or goat
  • it is transformed into a bio-factories for the production of foreign proteins of biopharmaceutical interest.
  • AAV vectors must contain an expression cassette that includes the DNA encoding the protein of interest, a sequence that encodes a secretion signal that may or may not be characteristic of the heterologous gene, a promoter that should not necessarily be specific to the glandular epithelium breast and a cutting and polyadenylation sequence.
  • the AAV vectors used for this purpose may be derived from human serotypes 1, 2, 3, 4, 5, 6, 7, 8 or 9. They may be chimeric vectors such as AAV-DJ or AAV-DJ / 8; or they can be vectors whose capsids have been modified through the use of combinatorial libraries to alter their tropism.
  • AAV vectors according to the method of the present invention allows obtaining high levels of recombinant proteins in milk for a period exceeding 100 days in immunocompetent animals.
  • the use of AAV vectors is an excellent option when it is desired to obtain large quantities of the protein of interest for the manufacturing, biomedical, livestock or aquaculture industry. These vectors have proven to be very stable in the EGM guaranteeing the expression in the milk of the recombinant protein for periods as prolonged as 4 months, in immunocompetent animals.
  • the AAV vectors of the present invention contain one or more expression cassette consisting of a promoter that may or may not be specific to EGM, a sequence encoding a protein of interest and a cutting and polyadenylation sequence. Constitutive promoters that can be heterologous for the transformed cell can be used for the construction of the expression cassette.
  • Suitable promoters include the immediate human cytomegalovirus (CMV) early promoter, the beta actin promoter (CAG), the elongation factor 1 alpha (EF1A) promoter, the SV40 virus early promoter (SV40), the promoter Phosphoglycerate kinase (PGK), the myosin heavy chain promoter (MyHc), the ubiquitin C promoter (UBC) and the Rous Sarcoma Virus (RSV) promoter.
  • CMV immediate human cytomegalovirus
  • CAG beta actin promoter
  • EF1A elongation factor 1 alpha
  • SV40 SV40 virus early promoter
  • PGK promoter Phosphoglycerate kinase
  • MyHc myosin heavy chain promoter
  • UHC ubiquitin C promoter
  • RSV Rous Sarcoma Virus
  • the promoters that naturally direct the expression of specific mammary gland genes could also be very useful, for example, aS
  • the sequence that codes for the protein of interest may be its complementary DNA that may or may not include introns to increase expression levels. It can also be used, if the capacity of the vector allows it, the genomic DNA that includes the introns of the gene of interest and has been shown to be capable of inducing expression levels higher than those obtained when complementary DNA is used.
  • any heterologous protein can be expressed in milk using the system proposed here. Particularly helpful could be the production of proteins with prophylactic or therapeutic value for humans and animals.
  • proteins that can be obtained by this route include, but are not limited to antigens (eg hepatitis B surface antigen), growth factors (eg, human growth hormone, epidermal growth factor, growth factor similar to insulin, granulocyte and macrophage colony stimulating factor, nerve growth factor, erythropoietin, etc.), coagulation factors (eg FVIII and FIX), Antibodies, cytokines (eg Interleukin 6 or Interleukin 2), ⁇ -antitrypsin, human serum albumin, ⁇ -globin, tissue activator of plasminogen, tumor suppressor proteins (eg P53), protein C, interferons.
  • antigens eg hepatitis B surface antigen
  • growth factors eg, human growth hormone, epidermal growth factor, growth factor similar to insulin, granulocyte and macro
  • Each heterologous protein produced according to this invention must be bound to a signal peptide that guarantees its secretion in milk.
  • the signal peptide may be characteristic of the heterologous protein when said protein is naturally secretable.
  • a heterologous signal peptide must be assembled in such a way that said peptide directs the secretion of the protein of interest.
  • the stability of messenger RNA is largely determined by the region located at the 3 'end of the gene.
  • This region must include a cutting and polyadenylation sequence that may be characteristic of the heterologous gene, although those derived from the globine genes, the bovine growth hormone gene, the thymidine kinase gene of the Herpes Simplex virus or from the early region of the SV40 virus.
  • the functionality of the expression cassette contained in AAV vectors can be tested in vitro by infecting a culture of mammary epithelial cells. Under infection, the protein of interest can be detected in the culture medium.
  • HC11 or KIM-2 mammary epithelial cells of murine origin, MAC-T of bovine origin, or GMGE of caprine origin could be particularly useful.
  • the method proposed in the present invention constitutes a solution to the increasingly growing production needs of protein constitution biopharmaceuticals.
  • the present invention allows large-scale production of proteins whose biological activity is closely related to complex post-translational processing and hence that require the biosynthetic machinery of the cells of higher organisms.
  • the method proposed here makes possible the production of said proteins in a quick, simple and economically feasible process.
  • the present invention allows to respond in a short time to changes in market needs, its application does not involve large technical requirements and its ability to react can be easily adjusted depending on the needs.
  • the present invention saves time and resources as an alternative for the study of post-translational modifications in the mammary gland of different species.
  • the coding sequence for the study protein is contained in an AAV vector, it can be transferred to the mammary gland of a wide range of species, thus allowing the study of the differential modifications that the same protein undergoes depending on the species where it is produced.
  • AAV vector construction was performed using the AAV-DJ He / per Free Expression System marketed by the company Cell Biolabs.
  • the helper plasmids pAAV-DJ (which supplies the Rec genes of AAV2 and AAV DJ Cap) and pHelper (which supplies the genes E2A, E4, and VA adenovirus RNA) in trans were used in trans.
  • plasmid pAAV-MCS was used, which contains and an immediate cytomegalovirus tempane promoter succeeded from a human beta-globin intron, a multiple cloning site, and a cutting and polyadenylation sequence.
  • the entire expression cassette is flanked by the repeated and inverted sequences of AAV.
  • the chemical sequence generated the coding sequence for a fusion protein composed of the human erythropoyectin sequence linked at its c-terminal end to the Fe regions of human inmuglobulin G (IgG).
  • the molecule encoded by this gene was called EPO-Fc (Fig. IB).
  • the gene coding for EPO-Fc was cloned between the Cia I and Xho I sites of the pAAV-MCS vector.
  • the resulting plasmid was called pAAV-EPOFc (Fig. 1A).
  • the plasmids pAAV-EPOFc, pAAV-DJ and pHelper were co-transfected into 293AAV cells (Cell biolabs) at a confluence of 80%.
  • the transfection was performed in a 10-level cell factory (EasyFill Cell Factory 10, 6320 cm 2 , Nunc). Plasmids were used in a 1: 1: 1 ratio, and transfection was performed using Polyethyleneimine 25kDa (Sigma-Aldrich) according to the procedure previously described by Toledo et al in 2009. After 72 hours, the cells were harvested and resuspended in a final volume of 100 mi.
  • the cell rupture was performed by three rounds of freezing at -20 ° C, defrosting at 37 ° C and vortex.
  • the cell debris was removed by centrifugation at 3000 RPM for 10 minutes.
  • the culture supernatant was clarified by sequential filtration through a prefilter, a 5 micron filter, a 0.4 micron filter and a final sterile filtration through 0.2 microns.
  • the AAV-EPOFc vector titer was determined by using the QuickTiter TM AAV Quantitation Kit (Cell biolabs). The sample containing the virus was stored at -80 ° C until it was used.
  • Example 2 Expression of EPO-Fc protein and from AAV in mammary epithelial cell culture.
  • the GMGE goat mammary epithelial cells were infected (Sánchez, O; 2007). The cells were cultured in DMEM medium supplemented with EGF (10 ng / ml) and insulin (10 ⁇ / ⁇ ). When the cells reached 100% confluence, the AAV vector was added at a rate of 20 viral particles per cell. After 24 hours the growth medium was replaced by means supplemented with EGF, insulin and 2% SFB. After 72 hours the medium was harvested.
  • Proteins contained in one milliliter of medium were precipitated with trichloroacetic acid and resuspended in 40 ⁇ of H 2 0 of which 20 ⁇ were used for electrophoresis and subsequent western blot assay (Fig. 2).
  • Example 3 Expression of EPO-Fc protein and from AAV in the milk of mice.
  • mice Two experimental groups of 5 mice each were made. The mice of the C57 line on day 17 of gestation were infused with 100 ⁇ per mammary gland of a preparation containing, depending on the experimental group: (I) 10 x 9 viral particles / ml of the AAV-EPO-Fc vector; (II) lxlO 9 viral particles / ml of a control AAV vector (AAV-GFP) condifying the green fluorescent protein; (III) PBS.
  • AAV-GFP control AAV vector
  • the genetic transformation of the bitter mass of the mice treated with the control / reporter vector AAV-GFP was evidenced on day 19 of pregnancy by exposing breast tissue biopsies to the fluorescence microscope and observing the green color in the breast tissue, indicative of GFP protein expression (Fig. 3A).
  • Treated mice began milking from day 2 postpartum.
  • the collected milk was diluted 1 in 5 with separation buffer (10 mM Tris-HCI pH 8, 10 m CaCI2) and the caseins were separated from the serum by centrifugation at 4 ° C for 30 minutes at 15000 g.
  • the EPO-Fc content in the milk samples was detected by Western blot and quantified by ELISA (Abcam, Erythropoietin [EPO] Human ELISA Klt. Cat. Abl 19522) (Fig. 3B-C).
  • Example 4 Induction of lactation in goats.
  • Recombinant AAV vectors as vehicles for transferring genes to the mammary gland, can be used in virtually any mammalian species. However, ruminants are preferred due to their high milk production capacity. Animals can be used at an early stage of sexual maturity to which mammogenesis and lactation can be hormonally induced or animals in the natural lactation phase can be used. If the animals to be treated are, for example, goats, hormonal induction can be performed by supplying estradiol (0.25 mg / kg, im) and progesterone (0.75 mg kg, im) on days 1, 3, 5, 7, 9 , 11 and 13 while prednisolone (0.4 mg / kg, im) should be administered on days 14 to 16 with daily udder massages from day 5.
  • Example 5 Infusion of the AAV-EPOFc vector in the mammary gland of goats.
  • Three goats in active lactation phase were given intramuscularly lmg mg of diazepan to reduce stress during treatment.
  • the animals were milked extensively to remove as much milk as possible in the cisterns.
  • the mammary glands were rinsed three times by infusion with saline solution at 37 ° C and subsequent milking.
  • a peristaltic pump was used for the infusion process in the mammary gland.
  • One end of the tubing was placed inside the jar containing the saline solution (150 mM NaCI, 30 mM EGTA), while the other end was inserted into the mammary gland through the nipple canal.
  • the infusions were carried out slowly while they were simultaneously massaged in the infused udders. Milking to remove the saline solution was done manually. Both udder media were filled with PBS supplemented with 33 mM EGTA and containing a viral load of 10 9 viral particles / ml. Approximately 1 liter of the solution containing the AAV-EPOFc vector was administered to each goat. That is, each mammary gland received a total of 5 x 10 11 viral particles. After the infusion, the udder was massaged to facilitate the solution to be distributed homogeneously and reach all the ducts and alveoli. The infused solution was removed the next day by milking.
  • Example 6 Detection of EPO-Fc in the milk of goats infused with the AAVFc vector.
  • the milk of the animals infused with the AAV-EPOFc vector began to be collected by manual milking from day 2, and until day 105 post infusion. Most of the milk collected was stored at -70 ° C for subsequent protein purification, while small samples were used to detect and quantify the content of EPO-Fc in each batch.
  • the EPO-Fc detection in milk was performed as follows: A sample of 150 ⁇ milk were added four volumes of separation buffer (10 mM Tris-HCl, 10 mM CaCl) and centrifuged at 4 ° C for 30 minutes at 15000 g to separate whey from fat and caseins. The serum fraction was recovered and the proteins contained in 10 ⁇ were separated by SDS-PAGE on a 12.5% acrylamide gel.
  • the proteins were transferred to a polyvinylidene difluoride membrane filter (PVDF) and labeled with an HRP-conjugated anti-human IgG antibody.
  • the immunoreactive bands were visualized by the chemiluminescent system (ECL) of Amersham Pharmacia Biotech (Fig. 4A).
  • EPO-Fc was carried out by ELISA (Abcam, Erythropoietin [EPO] Human ELISA Kit. Cat. Abl 19522), obtaining an expression range of 0.8 to 0.27 g / L (Fig. 4B) .
  • the presence of EPO-Fc in the milk of the infused goats could be detected during the 100 days that the milk was being collected, by both methods, ELISA and western blot.

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Abstract

The invention relates to a method that enables the production of recombinant proteins in the milk of non-transgenic mammals. The method is based on the direct transfer of the genetic information of interest to the cells forming the secretory mammary epithelium, by means of adeno-associated vectors. Using this method, it is possible to produce high levels of recombinant proteins in the milk of non-transgenic mammals during a complete lactation period. The method is scalable and enables sustained levels of expression to be achieved for periods of more than 100 days.

Description

METODO PARA LA PRODUCCION DE PROTEÍNAS RECOMBINANTES EN GLÁNDULA MAMARIA DE MAMÍFEROS MEDIANTE LA TRANSFORMACIÓN DEL EPITELIO GLANDULAR MAMARIO CON VECTORES ADENOASOCIADOS.  METHOD FOR THE PRODUCTION OF RECOMBINANT PROTEINS IN MAMMARY GLAND OF MAMMALS THROUGH THE TRANSFORMATION OF THE GLANDULAR MAMMARY EPITHELIUM WITH ADENO ASSOCIATED VECTORS.
SECTOR TECNICO TECHNICAL SECTOR
La tecnología que a continuación se presenta está destinada al sector biotecnología), específicamente a la producción de proteínas recombinantes, esta tecnología también está relacionada con el sector salud y farmacéutico. The technology presented below is intended for the biotechnology sector), specifically for the production of recombinant proteins, this technology is also related to the health and pharmaceutical sector.
TECNICA ANTERIOR PREVIOUS TECHNIQUE
El tratamiento de un gran número de enfermedades, requiere la administración de proteínas bioactivas. Estos biofármacos se pueden purificar a partir de la sangre y tejidos en un proceso que además de ser largo y costoso, encierra el riesgo de trasmisión de agentes infecciosos, tales como los causantes del SIDA y la hepatitis. The treatment of a large number of diseases requires the administration of bioactive proteins. These biopharmaceuticals can be purified from blood and tissues in a process that in addition to being long and expensive, entails the risk of transmission of infectious agents, such as the causes of AIDS and hepatitis.
El desarrollo de la tecnología del ADN recombinante ha permitido emplear bacterias y levaduras como sistemas hospederos para la producción de proteínas de interés biofarmacéutico en un proceso relativamente barato. Sin embargo, en muchas ocasiones la actividad biológica de dichas proteínas, se ve comprometida debido fundamentalmente al ineficiente procesamiento post-traduccional de estos sistemas de expresión, de aquí que la producción de un gran número de biofármacos proteicos, en su forma biológicamente activa, requiera de la maquinaria biosintética de células superiores. En este sentido los sistemas basados en baculovirus y cultivo de tejido de células de mamíferos han devenido en estrategias viables. Sin embargo la fermentación de células animales es un proceso caro y técnicamente exigente. El empleo de la glándula mamaria de animales transgénicos como sistema biorreactor se ha identificado como una solución potencial a los anteriores problemas. La ventaja fundamental de la transgénesis radica en el hecho de que, es posible crear una población transgénica a partir del animal fundador mediante reproducción natural. A pesar de las prometedoras perspectivas de esta tecnología, existe en la actualidad limitaciones que impiden su uso de una forma más generalizada. Particularmente cuando se trabaja con animales grandes se requiere mucho tiempo y recursos para producir un pequeño número de la generación inicial 0 (G0) transgénica. Solo una pequeña proporción de esta G0 transgénica expresa en su leche la proteína de interés a niveles razonables como para permitir la entrada en una fase comercial. Se requiere además un tiempo adicional largo para multiplicar mediante cruce las líneas G0 con altos niveles de expresión, hasta alcanzar una capacidad de producción adecuada que permita la entrada en el mercado. En este período de escalado también se corre el riesgo de que la progenie transgénica no exprese la proteína exógena a los mismos niveles que el fundador original. La clonación somática como una vía alternativa para la producción de animales transgénicos permite sobreponerse a algunas de las limitaciones anteriores sin embargo el alto costo así como la ineficacia de esta técnica atentan contra su práctica de una forma más rutinaria. El empleo de mamíferos transgénicos como biofábricas implica además la construcción de complejos casetes de expresión, donde el gen exógeno está unido a secuencias reguladoras que solo permitan su expresión en el epitelio glandular mamario durante la lactación. En muchas ocasiones estas secuencias reguladoras no funcionan eficientemente, provocando que pequeñas cantidades de proteína recombinante se sinteticen en otros tejidos, con un efecto detrimental para la salud del animal. The development of recombinant DNA technology has allowed the use of bacteria and yeasts as host systems for the production of proteins of biopharmaceutical interest in a relatively cheap process. However, in many cases the biological activity of these proteins is compromised mainly due to the inefficient post-translational processing of these expression systems, hence the production of a large number of protein biopharmaceuticals, in their biologically active form, requires of the biosynthetic machinery of superior cells. In this sense, systems based on baculovirus and tissue culture of mammalian cells have become viable strategies. However, fermentation of animal cells is an expensive and technically demanding process. The use of the mammary gland of transgenic animals as a bioreactor system has been identified as a potential solution to the above problems. The fundamental advantage of transgenesis lies in the fact that it is possible to create a transgenic population from the founding animal through natural reproduction. Despite the promising prospects for this technology, there is currently limitations that prevent its use in a more generalized way. Particularly when working with large animals, a lot of time and resources are required to produce a small number of the initial 0 (G0) transgenic generation. Only a small proportion of this transgenic G0 expresses in its milk the protein of interest at reasonable levels to allow entry into a commercial phase. It also takes a long additional time to multiply by crossing the G0 lines with high levels of expression, until reaching an adequate production capacity that allows entry into the market. In this period of escalation there is also a risk that the transgenic progeny does not express the exogenous protein at the same levels as the original founder. Somatic cloning as an alternative route for the production of transgenic animals allows some of the above limitations to be overcome, however, the high cost as well as the inefficiency of this technique undermine its practice in a more routine way. The use of transgenic mammals as biofactories also involves the construction of complex expression cassettes, where the exogenous gene is linked to regulatory sequences that only allow its expression in the mammary glandular epithelium during lactation. In many cases these regulatory sequences do not work efficiently, causing small amounts of recombinant protein to be synthesized in other tissues, with a detrimental effect on animal health.
La transferencia de genes directamente a las células del epitelio glandular mamario (EGM) en animales adultos resulta una estrategia muy prometedora. Esta tecnología podría no solo disminuir el costo de producción de biofármacos sino que también reducir considerablemente el tiempo necesario para la obtención de estos. La transformación in vivo del EGM se puede llevar a cabo en cualquier animal, independientemente de su fondo genético y con un mínimo de requerimientos técnicos.  The transfer of genes directly to mammary glandular epithelial cells (EGM) in adult animals is a very promising strategy. This technology could not only reduce the cost of producing biopharmaceuticals but also considerably reduce the time needed to obtain them. The in vivo transformation of EGM can be carried out in any animal, regardless of its genetic background and with a minimum of technical requirements.
Ya en trabajos tempranos (Archer y col.; Proc Nati Acad Sci U S A. 1994, 91(15):6840-4) se evaluó la factibilidad de emplear vectores retrovirales para transferir el gen de la hormona de crecimiento humano (hGH) a las células epiteliales mamarias de chivas. La hGH se pudo detectar en la leche, aunque a muy bajas concentraciones, y esta expresión cayó a niveles básales después del primer día. En los documentos de patentes US 5,215,904 y W0 99/43795 también se describen métodos donde se emplean retrovirus como vectores para la transformación del EGM. La endocitosis mediada por receptor en EP 725141 A4 y el empleo de complejos basados en policationes y/o lípidos en US 5,780,009 también se han descrito como alternativas para introducción de genes en el EGM. Aunque estos trabajos permiten obtener la proteína de interés en la leche de los animales tratados, hasta el momento las eficiencias reportadas son muy bajas y los niveles de proteínas obtenidos no exceden el orden de nanogramos por mililitro, lo cual constituye una limitante cuando se desea escalar estos métodos. Already in early work (Archer et al .; Proc Nati Acad Sci US A. 1994, 91 (15): 6840-4) the feasibility of using retroviral vectors to transfer the human growth hormone (hGH) gene to Chivas mammary epithelial cells. HGH could be detected in milk, although at very low concentrations, and this expression dropped to baseline levels after the first day. US 5,215,904 and W0 99/43795 also describe methods where retroviruses are used as vectors for the transformation of EGM. Endocytosis receptor-mediated in EP 725141 A4 and the use of complexes based on polycations and / or lipids in US 5,780,009 have also been described as alternatives for gene introduction into EGM. Although these works allow obtaining the protein of interest in the milk of treated animals, so far the reported efficiencies are very low and the levels of proteins obtained do not exceed the order of nanograms per milliliter, which constitutes a limitation when it is desired to scale these methods
El documento de patente WO 2004034780 A2 protege un procedimiento de transferencia de genes al epitelio glandular mamario que se basa en el empleo de vectores adenovirales como vehículo para la transferencia génica, a niveles superiores a 1 g/L La tecnología es eficiente. Sin embargo, la expresión de transgenes a partir de vectores adenovirales, generalmente se pierde en aproximadamente diez días. Esto se debe básicamente respuesta inmune que se levanta contra las células infectadas a causa de la inmunogenicidad de las proteínas virales expresadas en dichas células. Una limitante adicional de este proceso radica en que los animales no pueden ser reutilizados pues la respuesta inmune inducida por la primera administración viral, neutraliza totalmente cualquier proceso de transducción posterior. Patent document WO 2004034780 A2 protects a gene transfer procedure to the mammary glandular epithelium that is based on the use of adenoviral vectors as a vehicle for gene transfer, at levels greater than 1 g / L. The technology is efficient. However, the expression of transgenes from adenoviral vectors is generally lost in about ten days. This is basically due to an immune response that rises against infected cells because of the immunogenicity of viral proteins expressed in said cells. An additional limitation of this process is that animals cannot be reused because the immune response induced by the first viral administration totally neutralizes any subsequent transduction process.
Los virus adenoasociados (AAV) constituyen un grupo de virus de vertebrados, de la familia Parvoviridae y del género Dependovirus. El AAV es un virus muy simple de entre 45 y 60 nm de diámetro, que contiene un genoma de ADN lineal de simple cadena. El virus requiere la co-infección con adenovirus u otros para replicarse. El AAV está extendido en la población humana pero no está asociado con ninguna enfermedad conocida. La organización del genoma de los AAV es extremadamente simple, comprendiendo sólo dos genes: (i) rep, que codifica una familia de proteínas superpuestas involucradas en la replicación e integración; (ii) cap, que codifica una familia de tres proteínas estructurales víricas. Los extremos del genoma están compuestos por secuencias repetidas e invertidas (ITR) de cerca de 145 nucleótidos. Los AAV de tipo salvaje pueden integrar su ADN en el cromosoma del hospedador en ausencia del virus ayudante. Esta integración puede ocurrir con más frecuencia en regiones del cromosoma 19 y es dependiente de la presencia de la proteína rep.  Adeno-associated viruses (AAV) constitute a group of vertebrate viruses, of the Parvoviridae family and of the genus Dependovirus. AAV is a very simple virus between 45 and 60 nm in diameter, which contains a single stranded linear DNA genome. The virus requires co-infection with adenovirus or others to replicate. AAV is widespread in the human population but is not associated with any known disease. The organization of the AAV genome is extremely simple, comprising only two genes: (i) rep, which encodes a family of overlapping proteins involved in replication and integration; (ii) cap, which encodes a family of three viral structural proteins. The ends of the genome are composed of repeated and inverted sequences (ITR) of about 145 nucleotides. Wild-type AAVs can integrate their DNA into the host's chromosome in the absence of the helper virus. This integration may occur more frequently in regions of chromosome 19 and is dependent on the presence of the rep protein.
Los vectores de AAV contienen sólo las secuencias víricas ITR de origen viral. Entre estas secuencias se puede insertar el casetes de expresión deseado. El genoma de estos vectores no debe exceder el tamaño original de genoma viral (4.700 kb) para no afectar su eficiencia de empaquetamiento. Para la producción de AAV es necesario aportar en trans las proteínas rep y cap, lo cual se logra mediante el empleo de un virus ayudante, plásmidos, o una línea celular de complementación (Ponnazhagan, S; 2001). Las ventajas principales de los vectores basados en AAV, es que poseen un amplio tropismo, son muy pobremente inmunogénicos y son capaces de asegurar una expresión sostenida del transgén durante largos periodos de tiempo (vector Biolabs; 2007). AAV vectors contain only viral ITR viral sequences. Among these sequences the desired expression cassettes can be inserted. The genome of these vectors should not exceed the original viral genome size (4,700 kb) to Do not affect your packaging efficiency. For the production of AAV it is necessary to provide trans and cap proteins in trans, which is achieved through the use of a helper virus, plasmids, or a complementation cell line (Ponnazhagan, S; 2001). The main advantages of AAV-based vectors, are that they have a broad tropism, are very poorly immunogenic and are able to ensure sustained expression of the transgene for long periods of time (Biolabs vector; 2007).
BREVE DESCRIPCIÓN DE LAS FIGURAS BRIEF DESCRIPTION OF THE FIGURES
Figura 1: La figura muestra un esquema general del vector AAV-EPOFc (A) y el modelamiento de la molécula EPO-Fc diseñada (B)  Figure 1: The figure shows a general scheme of the AAV-EPOFc vector (A) and the modeling of the designed EPO-Fc molecule (B)
Figura 2: La figura muestra el análisis mediante un western blot de la proteína EPO-Fc expresada en cultivos celulares transducidos con el vector AAV-EPOFc utilizando un anticuerpo monoclonal específico contra EPO humana.  Figure 2: The figure shows the western blot analysis of the EPO-Fc protein expressed in cell cultures transduced with the AAV-EPOFc vector using a specific monoclonal antibody against human EPO.
Figura 3: La figura muestra una imagen del epitelio glandular mamario de ratón que fluorece fluorescente por acción del vector control AAV-GFP que induce la producción de la proteína reportea GFP en la células epiteliales transformadas (A); se muestra además la inmuno-identificación de la proteína recombinante EPO-Fc a partir de muestras de leche de ratones transducidos con el vector AAV-EPOFc, mediante un ensayo de western blot (B); se muestra la cuantificación de los niveles de expresión de EPO-Fc en la leche de los ratones transducidos con el vector AAV-EPOFc, mediante un inmunoensayo específico para EPO humana (ELISA).  Figure 3: The figure shows an image of the mouse mammary glandular epithelium that fluoresces fluorescently by action of the control vector AAV-GFP that induces the production of the GFP report protein in the transformed epithelial cells (A); immuno-identification of the recombinant EPO-Fc protein from milk samples of mice transduced with the AAV-EPOFc vector is also shown, by a western blot (B) assay; The quantification of EPO-Fc expression levels in the milk of mice transduced with the AAV-EPOFc vector is shown by a human EPO-specific immunoassay (ELISA).
Figura 4: La figura muestra la inmuno-dentificación de la expresión de EPO-Fc producida en la leche de cabras transducidas con el vector AAV-EPOFc. Se ensayaron cinco cabras durante un periodo de lactancia activa (100 día) y se colectaron muestras de leche con intervalos de 10 días por cada animal. Se cuantifico la cantidad de EPO-Fc recombinante producida por cada animal mediante ELISA (A), y se evalúo la presencia de la EPO-Fc en muestras de leche mediante un ensayo de western blot. DIVULGACION DE LA INVENCIÓN Figure 4: The figure shows the immuno-dentification of the expression of EPO-Fc produced in the milk of goats transduced with the AAV-EPOFc vector. Five goats were tested during an active lactation period (100 days) and milk samples were collected at 10 day intervals for each animal. The amount of recombinant EPO-Fc produced by each animal was quantified by ELISA (A), and the presence of EPO-Fc in milk samples was evaluated by a western blot assay. DISCLOSURE OF THE INVENTION
La presente invención corresponde a un método para la producción de biomoléculas recombinantes; empleándose la glándula mamaria como biorreactor y vectores AAV como vehículos para la transferencia génica. The present invention corresponds to a method for the production of recombinant biomolecules; the mammary gland being used as bioreactor and AAV vectors as vehicles for gene transfer.
Este método da solución a los problemas asociados a la generación de un animal transgénico, y además, permite solucionar las limitaciones de todas las estrategias previas de transducción del epitelio glandular mamario.  This method gives solution to the problems associated with the generation of a transgenic animal, and in addition, it allows solving the limitations of all the previous strategies of transduction of the mammary glandular epithelium.
El procedimiento general objeto de la presente invención costa de las siguientes etapas:  The general procedure object of the present invention costs the following steps:
1. Clonaje del gen de interés en el qenoma de un vector AAV: Implica la construcción de cásete de expresión para la proteína de interés flanqueado por las secuencias repetidas e invertidas de un vector AW. El procedimiento puede ser realizado empleando las herramientas convencionales de biología molecular o puede obtenerse mediante síntesis química. El tamaño total del constructo no debe exceder los 4,7 kb.  1. Cloning of the gene of interest in the qenoma of an AAV vector: It involves the construction of an expression cassette for the protein of interest flanked by the repeated and inverted sequences of an AW vector. The procedure can be performed using conventional molecular biology tools or can be obtained by chemical synthesis. The total size of the construct should not exceed 4.7 kb.
2. Generación de AAV: La generación de los vectores AAV puede ser realizada mediante cualquiera de los procedimientos disponibles en el estado del arte. Se puede emplear sistemas libres de virus ayudantes, basados en transfección transiente de un coctel de plásmidos donde uno de los plásmidos contiene el genoma que debe ser empaquetado, mientras que los restantes aportan en trans los genes rep y cap. También se puede emplear líneas celulares establemente productoras del vector AAV, o sistemas de producción en células de insecto, empleando baculovirus como vectores ayudantes.  2. Generation of AAV: The generation of AAV vectors can be performed by any of the procedures available in the state of the art. Assistive virus-free systems can be used, based on transient transfection of a plasmid cocktail where one of the plasmids contains the genome that must be packaged, while the rest provide trans and cap genes in trans. Stably producing cell lines of the AAV vector, or insect cell production systems, can also be employed, using baculovirus as helper vectors.
3. Instilación de los AAV en la glándula mamaria de un rumiante: Antes de la infusión que contiene los vectores AAV, el animal debe ser ordeñado para eliminar la leche contenida en las cisternas. La glándula mamaria es entonces infundida con una solución isoosmótica a través del canal del pezón hasta que la ubre esté llena, se le dan masajes a la ubre para hacer que la solución alcance la totalidad de los alvéolos y la solución es removida mediante ordeño. 3. Instilation of AAVs in the mammary gland of a ruminant: Before the infusion containing AAV vectors, the animal must be milked to remove milk contained in the cisterns. The mammary gland is then infused with an isoosmotic solution through the nipple canal until the udder is full, the udder is massaged to make the solution reach all the alveoli and the solution is removed by milking.
Este paso se debe repetir una o dos veces más para garantizar el total enjuague de la glándula mamaria y la distensión de los ductos y alvéolos. Como solución isoosmótica se puede emplear PBS, NaCI 0.9%, glucosa 5%, HBS o medio de cultivo de tejido. La infusión de la solución conteniendo las partículas virales se realiza directamente a través del canal del pezón. La concentración viral en la solución a infundir puede ser variable aunque concentraciones en el rango entre lxlO8 y lxlO10 pfu/mL son preferidas. This step should be repeated once or twice more to guarantee the total Rinse the mammary gland and distension of the ducts and alveoli. As an isoosmotic solution, PBS, 0.9% NaCI, 5% glucose, HBS or tissue culture medium can be used. The infusion of the solution containing the viral particles is carried out directly through the nipple canal. The viral concentration in the solution to be infused can be variable although concentrations in the range between 8 and lxlO lxlO 10 pfu / ml are preferred.
El volumen óptimo de la infusión puede variar en dependencia del tamaño de la ubre. Para realizar la infusión a través del canal del pezón se puede emplear una cánula acoplada a una jeringuilla o una bomba peristáltica. La infusión se debe hacer lentamente, y durante y después de la infusión se deben dar masajes para garantizar que la solución se distribuya homogéneamente y alcance la totalidad de las células epiteliales mamarias.  The optimal volume of the infusion may vary depending on the size of the udder. To infuse through the nipple canal, a cannula attached to a syringe or a peristaltic pump can be used. The infusion should be done slowly, and during and after the infusion massages should be given to ensure that the solution is distributed homogeneously and reaches all of the mammary epithelial cells.
El vector AAV puede infectar las células del epitelio glandular mamario usando como receptor primario las moléculas de heparan sulfato (HSPG), y como receptores secundarios a las integrina ανβ5 y al receptor del factor de crecimiento de fibroblastos tipo 1 (FGFR-1). Para asegurar el acceso del vector AAV a sus receptores, es aconsejable suplementar la solución de infusión con un agente quelante que facilite la ruptura temporal de las uniones estrechas entre las células que conforman el epitelio glandular mamario. Como agente quelante se puede usar ácido l,2-bis(2-aminofenoxi)etano-N,N,N',N'- tetraacético (BAPTA), ácido dimercaptosuccínico (DMSA), ácido dietilentriamino pentaacético (DTPA), ácido 2,3-dimercapto-l-propanosulfónico (DMPS), ácido nitrilo tri-acético (NTA), ácido de etileno glicol tetraacético (EGTA), dimercaprol (INN) o, ácido etilen diamino tetraacético (EDTA). The AAV vector can infect mammary glandular epithelial cells using heparan sulfate molecules (HSPG) as the primary receptor, and as secondary receptors to integrins α ν β 5 and fibroblast growth factor receptor type 1 (FGFR-1 ). To ensure access of the AAV vector to its receptors, it is advisable to supplement the infusion solution with a chelating agent that facilitates the temporary rupture of the tight junctions between the cells that make up the mammary glandular epithelium. As chelating agent, l, 2-bis (2-aminophenoxy) ethane-N, N, N ', N'-tetraacetic acid (BAPTA), dimercaptosuccinic acid (DMSA), diethylenetriamine pentaacetic acid (DTPA), acid 2, can be used 3-dimercapto-l-propanesulfonic acid (DMPS), tri-acetic nitrile acid (NTA), ethylene glycol tetraacetic acid (EGTA), dimercaprol (INN) or, ethylene diamine tetraacetic acid (EDTA).
Colecta de la leche: La leche, a partir de los animales tratados, se puede obtener mediante los métodos convencionales de ordeño manual o mecanizado. Se estará colectando desde el segundo día de realizado el procedimiento hasta que los niveles de expresión sean atractivos desde el punto de vista industrial (usualmente 100-140 días).  Milk collection: Milk, from treated animals, can be obtained by conventional methods of manual or mechanized milking. It will be collected from the second day of the procedure until the levels of expression are attractive from an industrial point of view (usually 100-140 days).
Purificación de la proteína de interés a partir de la leche: La mayor parte de la leche es almacenada a -20°C mientras que pequeñas muestras son empleadas para detectar y cuantificar la proteína de interés con el uso de técnicas comúnmente conocidas (Ej. ELISA, Western blot o actividad biológica). Los sueros que contengan cantidades apreciables de la proteína de interés son mezclados y empleados como materia prima activa para la purificación de la proteína heteróloga. El proceso de purificación puede variar considerablemente en dependencia de la proteína en cuestión. Purification of the protein of interest from milk: Most of the milk is stored at -20 ° C while small samples are used to detect and quantify the protein of interest with the use of techniques commonly known (eg ELISA, Western blot or biological activity). Serums containing appreciable amounts of the protein of interest are mixed and used as active raw material for purification of the heterologous protein. The purification process can vary considerably depending on the protein in question.
El procedimiento es robusto, garantizando altos niveles de expresión de proteínas recombinantes y asegurando una producción sostenida por periodos mayores a los 100 días. The procedure is robust, guaranteeing high levels of recombinant protein expression and ensuring sustained production for periods longer than 100 days.
El método de la presente invención involucra la infusión de una solución que contiene los vectores AAV directamente a través del canal del pezón. Los animales deben ser preferentemente rumiantes (Ej. bovinos, ovinos o caprinos). Los animales pueden ser sometidos a una inducción hormonal de mamogénesis y lactación o se pueden emplear animales que se encuentren en fase natural de lactación. En virtud de la infección, los transgenes son transferidos al interior de las células secretoras mamarias donde son transcriptos y donde ocurre la traducción y los procesos post- traduccionales que requiere la proteína de interés antes de ser secretada en la leche. Mediante el ordeño del animal (Ej. vaca, oveja o cabra) este se transforma en una biofactoría para la producción de proteínas foráneas de interés biofarmacéutico. Los vectores AAV deben contener un cásete de expresión que incluya el ADN codificante para la proteína de interés, una secuencia que codifique para una señal de secreción que puede o no ser propia del gen heterólogo, un promotor que no necesariamente debe ser específico del epitelio glandular mamario y una secuencia de corte y poliadenilación.  The method of the present invention involves the infusion of a solution containing AAV vectors directly through the nipple channel. Animals should preferably be ruminants (eg bovines, sheep or goats). Animals can be subjected to a hormonal induction of mammogenesis and lactation or animals that are in the natural phase of lactation can be used. By virtue of the infection, the transgenes are transferred into the mammary secretory cells where they are transcribed and where the translation and post-translational processes that the protein of interest requires before being secreted in the milk. By milking the animal (eg cow, sheep or goat) it is transformed into a bio-factories for the production of foreign proteins of biopharmaceutical interest. AAV vectors must contain an expression cassette that includes the DNA encoding the protein of interest, a sequence that encodes a secretion signal that may or may not be characteristic of the heterologous gene, a promoter that should not necessarily be specific to the glandular epithelium breast and a cutting and polyadenylation sequence.
Los vectores AAV empleados para este propósito pueden derivarse de los serotipos humanos 1, 2, 3, 4, 5, 6, 7, 8 o 9. Pueden ser vectores quiméricos como los AAV-DJ o AAV-DJ/8; o pueden ser vectores cuyas cápsides ha sido modificada mediante el empleo de bibliotecas combinatorias para alterar su tropismo.  The AAV vectors used for this purpose may be derived from human serotypes 1, 2, 3, 4, 5, 6, 7, 8 or 9. They may be chimeric vectors such as AAV-DJ or AAV-DJ / 8; or they can be vectors whose capsids have been modified through the use of combinatorial libraries to alter their tropism.
El empleo de vectores AAV según el método de la presente invención permite obtener altos niveles de proteínas recombinantes en la leche durante un periodo superior a los 100 días en animales inmunocompetentes. El uso de vectores AAV constituye una excelente opción cuando se desea obtener grandes cantidades de la proteína de interés para la industria manufacturera, biomédica, pecuaria o acuicola. Estos vectores han demostrados ser muy estables en el EGM garantizando la expresión en la leche de la proteína recombinante durante períodos tan prolongados como 4 meses, en animales inmunocompetentes. The use of AAV vectors according to the method of the present invention allows obtaining high levels of recombinant proteins in milk for a period exceeding 100 days in immunocompetent animals. The use of AAV vectors is an excellent option when it is desired to obtain large quantities of the protein of interest for the manufacturing, biomedical, livestock or aquaculture industry. These vectors have proven to be very stable in the EGM guaranteeing the expression in the milk of the recombinant protein for periods as prolonged as 4 months, in immunocompetent animals.
Los vectores AAV de la presente invención contienen uno o más cásete de expresión que consta de un promotor que puede o no ser específico del EGM, una secuencia que codifica para una proteína de interés y una secuencia de corte y poliadenilación. Para la construcción del cásete de expresión se pueden emplear promotores constitutivos que pueden ser heterólogos para la célula transformada. Ejemplos de promotores adecuados incluyen el promotor inmediato temprano de citomegalovirus humano (CMV), el promotor de beta actina (CAG), el promotor del factor de elongación 1 alfa (EF1A), el promotor temprano del virus SV40 (SV40), el promotor de fosfoglicerato kinasa (PGK), el promotor de la cadena pesada de la miosina (MyHc), el promotor de ubiquitina C (UBC) y el promotor de Rous Sarcoma Virus (RSV). De gran utilidad también podrían ser los promotores que de forma natural dirigen la expresión de genes específicos de la glándula mamaria, se pueden emplear por ejemplo promotores de aSl-caseína, aS2-caseína, β-lactoglobulina, κ-caseína, β- caseína, promotores de la proteína acídica del suero y de a-lactoalbúmina.  The AAV vectors of the present invention contain one or more expression cassette consisting of a promoter that may or may not be specific to EGM, a sequence encoding a protein of interest and a cutting and polyadenylation sequence. Constitutive promoters that can be heterologous for the transformed cell can be used for the construction of the expression cassette. Examples of suitable promoters include the immediate human cytomegalovirus (CMV) early promoter, the beta actin promoter (CAG), the elongation factor 1 alpha (EF1A) promoter, the SV40 virus early promoter (SV40), the promoter Phosphoglycerate kinase (PGK), the myosin heavy chain promoter (MyHc), the ubiquitin C promoter (UBC) and the Rous Sarcoma Virus (RSV) promoter. The promoters that naturally direct the expression of specific mammary gland genes could also be very useful, for example, aSl-casein, aS2-casein, β-lactoglobulin, κ-casein, β-casein promoters can be used. promoters of serum acidic protein and α-lactalbumin.
La secuencia que codifica para la proteína de interés puede ser su ADN complementario que puede o no incluir intrones para incrementar los niveles de expresión. También se puede utilizar, si la capacidad del vector lo permite, el ADN genómico que incluye los intrones propios del gen de interés y se ha demostrado que es capaz de inducir niveles de expresión superiores a los obtenidos cuando se emplea ADN complementario.  The sequence that codes for the protein of interest may be its complementary DNA that may or may not include introns to increase expression levels. It can also be used, if the capacity of the vector allows it, the genomic DNA that includes the introns of the gene of interest and has been shown to be capable of inducing expression levels higher than those obtained when complementary DNA is used.
Virtualmente cualquier proteína heteróloga puede ser expresada en la leche empleando el sistema aquí propuesto. Particularmente provechosa podría ser la producción de proteínas con valor profiláctico o terapéutico para humanos y animales. Ejemplos de proteínas que se pueden obtener por esta vía incluyen, pero no están limitados a antígenos (Ej antígeno de superficie de la hepatitis B), factores de crecimiento (Ej. Hormona de crecimiento humano, factor de crecimiento epidérmico, factor de crecimiento similar a la insulina, factor estimulador de colonias de granulositos y macrófagos, factor de crecimiento nervioso, eritropoietina, etc), factores de coagulación (Ej. FVIII y FIX), Anticuerpos, citoquinas (Ej. Interleukina 6 o Interleukina 2), α-antitripsina, seroalbúmina humana, β-globina, activador tisular del plasminógeno, proteínas supresoras de tumores (Ej. P53), proteína C, ¡nterferones. Cada proteína heteróloga producida acorde a esta invención debe estar unida a un péptido señal que garantice su secreción en la leche. El péptido señal puede ser propio de la proteína heteróloga cuando dicha proteína es naturalmente secretable. Cuando la proteína que se desea producir no es una proteína secretable, entonces en la construcción genética se debe ensamblar un péptido señal heterólogo de forma tal que dicho péptido dirija la secreción de la proteína de interés. La estabilidad del ARN mensajero está determinada en gran medida por la región ubicada en el extremo 3' del gen. Esta región debe incluir una secuencia de corte y poliadenilación que puede ser propia del gen heterólogo, aunque comúnmente se emplean aquellas derivadas de los genes de globinas, del gen de la hormona de crecimiento bovina, del gen de timidina kinasa del virus Herpes Simple o de la región temprana del virus SV40. Virtually any heterologous protein can be expressed in milk using the system proposed here. Particularly helpful could be the production of proteins with prophylactic or therapeutic value for humans and animals. Examples of proteins that can be obtained by this route include, but are not limited to antigens (eg hepatitis B surface antigen), growth factors (eg, human growth hormone, epidermal growth factor, growth factor similar to insulin, granulocyte and macrophage colony stimulating factor, nerve growth factor, erythropoietin, etc.), coagulation factors (eg FVIII and FIX), Antibodies, cytokines (eg Interleukin 6 or Interleukin 2), α-antitrypsin, human serum albumin, β-globin, tissue activator of plasminogen, tumor suppressor proteins (eg P53), protein C, interferons. Each heterologous protein produced according to this invention must be bound to a signal peptide that guarantees its secretion in milk. The signal peptide may be characteristic of the heterologous protein when said protein is naturally secretable. When the protein to be produced is not a secretable protein, then in the genetic construction a heterologous signal peptide must be assembled in such a way that said peptide directs the secretion of the protein of interest. The stability of messenger RNA is largely determined by the region located at the 3 'end of the gene. This region must include a cutting and polyadenylation sequence that may be characteristic of the heterologous gene, although those derived from the globine genes, the bovine growth hormone gene, the thymidine kinase gene of the Herpes Simplex virus or from the early region of the SV40 virus.
La funcionalidad del cásete de expresión contenido en los vectores AAV puede ser ensayada in vitro mediante la infección de un cultivo de células epiteliales mamarias. En virtud de la infección, la proteína de interés puede ser detectada en el medio de cultivo. En este sentido podrían ser particularmente útiles las líneas de células epiteliales mamarias HC11 o KIM-2 de origen murino, MAC-T de origen bovino, o GMGE de origen caprino.  The functionality of the expression cassette contained in AAV vectors can be tested in vitro by infecting a culture of mammary epithelial cells. Under infection, the protein of interest can be detected in the culture medium. In this regard, HC11 or KIM-2 mammary epithelial cells of murine origin, MAC-T of bovine origin, or GMGE of caprine origin could be particularly useful.
El método propuesto en la presente invención constituye una solución a las necesidades cada vez más crecientes de producción de biofármacos de constitución proteica. Específicamente la presente invención permite la producción a gran escala de proteínas cuya actividad biológica está estrechamente relacionada con complejos procesamientos post-traduccionales y de aquí que requieran de la maquinaria biosintética de las células de organismos superiores. El método aquí propuesto hace posible la producción de dichas proteínas en un proceso rápido, sencillo y económicamente factible. En este sentido la presente invención permite dar respuesta en breve tiempo a los cambios en las necesidades del mercado, su aplicación no involucra grandes requerimientos técnicos y su capacidad de reacción puede ser fácilmente ajustada en dependencia de las necesidades.  The method proposed in the present invention constitutes a solution to the increasingly growing production needs of protein constitution biopharmaceuticals. Specifically, the present invention allows large-scale production of proteins whose biological activity is closely related to complex post-translational processing and hence that require the biosynthetic machinery of the cells of higher organisms. The method proposed here makes possible the production of said proteins in a quick, simple and economically feasible process. In this sense, the present invention allows to respond in a short time to changes in market needs, its application does not involve large technical requirements and its ability to react can be easily adjusted depending on the needs.
Adicionalmente la presente invención permite salvar tiempo y recursos como una alternativa para el estudio de las modificaciones post traduccionales en la glándula mamaria de diferentes especies. Teniendo en cuenta que la secuencia codificante para la proteína de estudio está contenida en un vector AAV, este puede ser transferido a la glándula mamaria de un amplio rango de especies, permitiendo de esta forma el estudio de las modificaciones diferenciales que sufre una misma proteína en dependencia de la especie donde se produzca. Additionally, the present invention saves time and resources as an alternative for the study of post-translational modifications in the mammary gland of different species. Given that the coding sequence for the study protein is contained in an AAV vector, it can be transferred to the mammary gland of a wide range of species, thus allowing the study of the differential modifications that the same protein undergoes depending on the species where it is produced.
EJEMPLOS DE APLICACIÓN: APPLICATION EXAMPLES:
Ejemplo 1: Generación del Vector AAV Example 1: AAV Vector Generation
La construcción de vector AAV se realizó empleando el AAV-DJ He/per Free Expression System comercializado por la empresa Cell Biolabs. Se usaron los plásmidos ayudantes pAAV-DJ (que suministra en trans los genes Rec de AAV2 y AAV DJ Cap) y pHelper (que suministra en trans los genes E2A, E4, y VA RNA de adenovirus). Como vector de expresión, se empleó el plasmidio pAAV-MCS, que contiene y un promotor inmediato tempano de citomegalovirus sucedido de un intrón de beta-globina humana, un sitio múltiple de clonaje, y una secuencia de corte y poliadenilación. Todo el cásete de expresión está flanqueado por las secuencias repetidas e invertidas de AAV. AAV vector construction was performed using the AAV-DJ He / per Free Expression System marketed by the company Cell Biolabs. The helper plasmids pAAV-DJ (which supplies the Rec genes of AAV2 and AAV DJ Cap) and pHelper (which supplies the genes E2A, E4, and VA adenovirus RNA) in trans were used in trans. As an expression vector, plasmid pAAV-MCS was used, which contains and an immediate cytomegalovirus tempane promoter succeeded from a human beta-globin intron, a multiple cloning site, and a cutting and polyadenylation sequence. The entire expression cassette is flanked by the repeated and inverted sequences of AAV.
Mediante síntesis química se generó la secuencia codificante para una proteína fusión compuesta por la secuencia de eritropoyectina humana unida en su extremo c- terminal a las región Fe de la inmuglobulina G humana (IgG). La molécula codificada por este gen fue denominada EPO-Fc (Fig. IB). El gen codificante para la EPO-Fc se clonó entre los sitios Cía I y Xho I del vector pAAV-MCS. El plasmidio resultante se denominó pAAV-EPOFc (Fig. 1A).  The chemical sequence generated the coding sequence for a fusion protein composed of the human erythropoyectin sequence linked at its c-terminal end to the Fe regions of human inmuglobulin G (IgG). The molecule encoded by this gene was called EPO-Fc (Fig. IB). The gene coding for EPO-Fc was cloned between the Cia I and Xho I sites of the pAAV-MCS vector. The resulting plasmid was called pAAV-EPOFc (Fig. 1A).
Para la generación el vector adenoasociados AAV-EPOFc, los plásmidos pAAV- EPOFc, pAAV-DJ y pHelper fueron co-transfectados en células 293AAV (Cell biolabs) a una confluencia del 80%. La transfección se realizó en una factoría celular de 10 niveles (EasyFill Cell Factory 10, 6320 cm2, Nunc). Los plásmidos fueron usado en una relación 1:1:1, y la transfección se realizó empleando Polietilenimina 25kDa (Sigma- Aldrich) acorde al procedimiento previamente descrito por Toledo y colaboradores en 2009. Pasadas 72 horas, la células fueron cosechadas y resuspendidas en un volumen final de 100 mi. Se realizó la ruptura celular mediante tres rondas de congelación a - 20°C, descongelación a 37°C y vortex. Los restos celulares se removieron mediante centrifugación a 3000 RPM por 10 minutos. El sobrenadante de cultivo se clarificó mediante filtración secuencial a través de un prefiltro, un filtro de 5 mieras, un filtro de 0,4 mieras y una filtración estéril final a través 0,2 mieras. El título del vector AAV- EPOFc se determinó mediante el empleo del QuickTiter™ AAV Quantitation Kit (Cell biolabs). La muestra que contiene el virus fue almacenada a -80°C hasta el momento de su uso. For generation of the adeno-associated AAV-EPOFc vector, the plasmids pAAV-EPOFc, pAAV-DJ and pHelper were co-transfected into 293AAV cells (Cell biolabs) at a confluence of 80%. The transfection was performed in a 10-level cell factory (EasyFill Cell Factory 10, 6320 cm 2 , Nunc). Plasmids were used in a 1: 1: 1 ratio, and transfection was performed using Polyethyleneimine 25kDa (Sigma-Aldrich) according to the procedure previously described by Toledo et al in 2009. After 72 hours, the cells were harvested and resuspended in a final volume of 100 mi. The cell rupture was performed by three rounds of freezing at -20 ° C, defrosting at 37 ° C and vortex. The cell debris was removed by centrifugation at 3000 RPM for 10 minutes. The culture supernatant was clarified by sequential filtration through a prefilter, a 5 micron filter, a 0.4 micron filter and a final sterile filtration through 0.2 microns. The AAV-EPOFc vector titer was determined by using the QuickTiter ™ AAV Quantitation Kit (Cell biolabs). The sample containing the virus was stored at -80 ° C until it was used.
Ejemplo 2: Expresión de proteína EPO-Fc y a partir de AAV en cultivo de células epiteliales mamarias. Example 2: Expression of EPO-Fc protein and from AAV in mammary epithelial cell culture.
Para comprobar el correcto funcionamiento del casetes de expresión de EPO-Fc en el vector AAV-EPOFc, se infectaron las células epiteliales mamarias de cabra GMGE (Sánchez, O; 2007). Las células se cultivaron en medio DMEM suplementado con EGF (10 ng/ml) e insulina (10 μς/ιηΙ). Cuando las células alcanzaron un 100 % de confluencia se les adicionó el vector AAV a razón de 20 partículas víricas por célula. Al cabo de 24 horas el medio de crecimiento fue reemplazado por medio suplementado con EGF, insulina y 2% de SFB. Pasadas 72 horas el medio se cosechó. Las proteínas contenidas en un mililitro de medio fueron precipitadas con ácido tricloroacético y resuspendidas en 40 μΙ de H20 de los cuales 20 μΙ se utilizaron para electroforesis y subsecuente ensayo de western blot (Fig. 2). To verify the correct functioning of the EPO-Fc expression cassettes in the AAV-EPOFc vector, the GMGE goat mammary epithelial cells were infected (Sánchez, O; 2007). The cells were cultured in DMEM medium supplemented with EGF (10 ng / ml) and insulin (10 μς / ιηΙ). When the cells reached 100% confluence, the AAV vector was added at a rate of 20 viral particles per cell. After 24 hours the growth medium was replaced by means supplemented with EGF, insulin and 2% SFB. After 72 hours the medium was harvested. Proteins contained in one milliliter of medium were precipitated with trichloroacetic acid and resuspended in 40 μΙ of H 2 0 of which 20 μΙ were used for electrophoresis and subsequent western blot assay (Fig. 2).
Ejemplo 3: Expresión de proteína EPO-Fc y a partir de AAV en la leche de ratones. Example 3: Expression of EPO-Fc protein and from AAV in the milk of mice.
La capacidad del vector AAV para infectar el EGM y promover la subsecuente secreción en la leche de la proteína de interés fue ensayada en ratones. Se hicieron dos grupos experimentales de 5 ratonas cada uno. A las ratonas de la línea C57 en el día 17 de gestación se les infundieron 100 μΙ por glándula mamaria de una preparación que contenía en dependencia del grupo experimental: (I) lxlO9 partículas virales/ml del vector AAV-EPO-Fc; (II) lxlO9 partículas virales/ml de un vector AAV control (AAV- GFP) condificante para la proteína fluorescente verde; (III) PBS. La transformación genética de la masa amaría de los ratones tratados con el vector control/reportero AAV-GFP se evidenció en el día 19 de gestación al exponer biopsias de tejido mamario al microscopio de fluorescencia y observar el color verde en el tejido mamario, indicativo de la expresión de la proteína GFP (Fig. 3A). Los ratones tratados se comenzaron a ordeñar a partir del día 2 post parto. La leche colectada se diluyó 1 en 5 con buffer de separación (10 mM Tris-HCI pH 8, 10 m CaCI2) y las caseínas se separaron del suero mediante centrifugación a 4°C durante 30 minutos a 15000 g. El contenido de EPO-Fc en las muestras de leche fue detectado por Western blot y cuantificado por ELISA (Abcam, Erythropoietin [EPO] Human ELISA Klt. Cat. abl 19522) (Fig. 3B-C). The ability of the AAV vector to infect EGM and promote subsequent milk secretion of the protein of interest was tested in mice. Two experimental groups of 5 mice each were made. The mice of the C57 line on day 17 of gestation were infused with 100 μΙ per mammary gland of a preparation containing, depending on the experimental group: (I) 10 x 9 viral particles / ml of the AAV-EPO-Fc vector; (II) lxlO 9 viral particles / ml of a control AAV vector (AAV-GFP) condifying the green fluorescent protein; (III) PBS. The genetic transformation of the bitter mass of the mice treated with the control / reporter vector AAV-GFP was evidenced on day 19 of pregnancy by exposing breast tissue biopsies to the fluorescence microscope and observing the green color in the breast tissue, indicative of GFP protein expression (Fig. 3A). Treated mice began milking from day 2 postpartum. The collected milk was diluted 1 in 5 with separation buffer (10 mM Tris-HCI pH 8, 10 m CaCI2) and the caseins were separated from the serum by centrifugation at 4 ° C for 30 minutes at 15000 g. The EPO-Fc content in the milk samples was detected by Western blot and quantified by ELISA (Abcam, Erythropoietin [EPO] Human ELISA Klt. Cat. Abl 19522) (Fig. 3B-C).
Ejemplo 4: Inducción de lactación en cabras. Example 4: Induction of lactation in goats.
Los vectores AAV recombinantes, como vehículos para transferir genes a la glándula mamaria, pueden ser empleados virtualmente en cualquier especie de mamíferos. Sin embargo, los rumiantes son preferidos debido a su alta capacidad de producción de leche. Se pueden usar animales en una etapa temprana de la madurez sexual a los cuales se les puede inducir hormonalmente la mamogénesis y lactación o se pueden emplear animales en fase natural de lactación. Si los animales a tratar son por ejemplo cabras, la inducción hormonal se puede realizar mediante el suministro de estradiol (0.25 mg/kg, i.m) y progesterona (0.75 mg kg, i.m.) en los días 1, 3, 5, 7, 9, 11 y 13 mientras que prednisolona (0.4 mg/kg, i.m) debe ser administrada en los días 14 al 16 con masajes diarios en la ubre a partir del día 5.  Recombinant AAV vectors, as vehicles for transferring genes to the mammary gland, can be used in virtually any mammalian species. However, ruminants are preferred due to their high milk production capacity. Animals can be used at an early stage of sexual maturity to which mammogenesis and lactation can be hormonally induced or animals in the natural lactation phase can be used. If the animals to be treated are, for example, goats, hormonal induction can be performed by supplying estradiol (0.25 mg / kg, im) and progesterone (0.75 mg kg, im) on days 1, 3, 5, 7, 9 , 11 and 13 while prednisolone (0.4 mg / kg, im) should be administered on days 14 to 16 with daily udder massages from day 5.
Ejemplo 5: Infusión del vector AAV-EPOFc en la glándula mamaria de cabras. Example 5: Infusion of the AAV-EPOFc vector in the mammary gland of goats.
A tres cabras en fase de lactación activa (aproximadamente 1 litro de leche diario) se les administró intramuscularmente lOmg de diazepán para disminuir el estrés durante el tratamiento. Los animales fueron ordeñados extensivamente para eliminar la mayor cantidad posible de leche en las cisternas. Las glándulas mamarias fueron enjuagadas tres veces mediante la infusión con solución salina a 37°C y posterior ordeño. Para el proceso de infusión en la glándula mamaria, se empleó una bomba peristáltica. Uno de los extremos de la tubería se colocó dentro del frasco conteniendo la solución salina (150 mM de NaCI, 30 mM de EGTA), mientras que el otro extremo se insertó en la glándula mamaria a través del canal de pezón. Las infusiones se realizaron lentamente mientras que de forma simultánea se les daban masajes en las ubres infundidas. Los ordeños para eliminar la solución salina se realizaron de forma manual. Ambos medios de la ubre fueron llenados con PBS suplementado con 33 mM de EGTA y conteniendo una carga viral de 109 partículas virales/ml. A cada cabra se le administró aproximadamente 1 litro de la solución conteniendo el vector AAV-EPOFc. Es decir, cada glándula mamaria recibió un total de 5 x 1011 partículas víricas. Después de la infusión se les dieron masajes a la ubre para facilitar que la solución se distribuyera homogéneamente y alcanzara la totalidad de ductos y alvéolos. La solución infundida se removió al siguiente día mediante ordeño. Three goats in active lactation phase (approximately 1 liter of milk daily) were given intramuscularly lmg mg of diazepan to reduce stress during treatment. The animals were milked extensively to remove as much milk as possible in the cisterns. The mammary glands were rinsed three times by infusion with saline solution at 37 ° C and subsequent milking. For the infusion process in the mammary gland, a peristaltic pump was used. One end of the tubing was placed inside the jar containing the saline solution (150 mM NaCI, 30 mM EGTA), while the other end was inserted into the mammary gland through the nipple canal. The infusions were carried out slowly while they were simultaneously massaged in the infused udders. Milking to remove the saline solution was done manually. Both udder media were filled with PBS supplemented with 33 mM EGTA and containing a viral load of 10 9 viral particles / ml. Approximately 1 liter of the solution containing the AAV-EPOFc vector was administered to each goat. That is, each mammary gland received a total of 5 x 10 11 viral particles. After the infusion, the udder was massaged to facilitate the solution to be distributed homogeneously and reach all the ducts and alveoli. The infused solution was removed the next day by milking.
Ejemplo 6: Detección de EPO-Fc en la leche de cabras infundidas con el vector AAVFc. Example 6: Detection of EPO-Fc in the milk of goats infused with the AAVFc vector.
La leche de los animales infundidos con el vector AAV-EPOFc se comenzó a colectar mediante ordeño manual a partir del día 2, y hasta el día 105 post infusión. La mayor parte de la leche colectada se almacenó a -70°C para la posterior purificación de la proteína, mientras que pequeñas muestras fueron usadas para detectar y cuantificar el contenido de EPO-Fc en cada uno de los lotes. La detección EPO-Fc en la leche se realizó como sigue: A muestras de 150 μΙ de leche se les adicionaron cuatro volúmenes de buffer de separación (10 mM Tris-HCI, 10 mM CaCI) y se centrifugaron a 4o C durante 30 minutos a 15000 g para separar el suero de la grasa y las caseínas. La fracción del suero fue recobrada y las proteínas contenidas en 10 μΙ fueron separadas mediante SDS-PAGE en un gel de acrilamida al 12.5%. Las proteínas se transfirieron a un filtro de membrana de difluoruro de polivinilideno (PVDF) y marcadas con un anticuerpo anti-human IgG conjugado con HRP. Las bandas inmunorreactivas fueron visualizadas mediante el sistema quimioluminiscente (ECL) de Amersham Pharmacia Biotech (Fig. 4A). La cuantificación de EPO-Fc se llevó a cabo mediante ELISA (Abcam, Erythropoietin [EPO] Human ELISA Kit. Cat. abl 19522), obteniéndose un rango de expresión de 0,8 a 0,27 g/L (Fig. 4B). La presencia de EPO-Fc en la leche de las cabras infundidas pudo ser detectada durante los 100 días que se estuvo colectando la leche, por ambos métodos, ELISA y western blot. The milk of the animals infused with the AAV-EPOFc vector began to be collected by manual milking from day 2, and until day 105 post infusion. Most of the milk collected was stored at -70 ° C for subsequent protein purification, while small samples were used to detect and quantify the content of EPO-Fc in each batch. The EPO-Fc detection in milk was performed as follows: A sample of 150 μΙ milk were added four volumes of separation buffer (10 mM Tris-HCl, 10 mM CaCl) and centrifuged at 4 ° C for 30 minutes at 15000 g to separate whey from fat and caseins. The serum fraction was recovered and the proteins contained in 10 μΙ were separated by SDS-PAGE on a 12.5% acrylamide gel. The proteins were transferred to a polyvinylidene difluoride membrane filter (PVDF) and labeled with an HRP-conjugated anti-human IgG antibody. The immunoreactive bands were visualized by the chemiluminescent system (ECL) of Amersham Pharmacia Biotech (Fig. 4A). The quantification of EPO-Fc was carried out by ELISA (Abcam, Erythropoietin [EPO] Human ELISA Kit. Cat. Abl 19522), obtaining an expression range of 0.8 to 0.27 g / L (Fig. 4B) . The presence of EPO-Fc in the milk of the infused goats could be detected during the 100 days that the milk was being collected, by both methods, ELISA and western blot.

Claims

REIVINDICACIONES
1. Un método para la producción de proteínas recombinantes en la leche de mamíferos no humanos mediante la transformación del epitelio glandular mamario con vectores adenoasociados, CARACTERIZADO porque comprende los siguientes pasos: a. Clonaje del gen de interés en el genoma de un vector AAV; 1. A method for the production of recombinant proteins in the milk of non-human mammals by transforming the mammary glandular epithelium with adeno-associated vectors, CHARACTERIZED because it comprises the following steps: a. Cloning of the gene of interest in the genome of an AAV vector;
b. Generación de AAV recombinantes ¡n vitro;  b. In vitro recombinant AAV generation;
c. Instilación de una solución conteniendo el vector AAV en la glándula mamaria de un rumiante;  C. Instilation of a solution containing the AAV vector in the mammary gland of a ruminant;
d. Colecta de la leche; y  d. Milk collection; Y
e. Purificación de la proteína de interés a partir de la leche.  and. Purification of the protein of interest from milk.
2. El método para la producción de proteínas recombinantes, según reivindicación 1, CARACTERIZADO porque el gen interés puede codificar para una molécula de interés para la industria manufacturera, biomédica, pecuaria o acuícola. 2. The method for the production of recombinant proteins, according to claim 1, CHARACTERIZED because the gene of interest can code for a molecule of interest for the manufacturing, biomedical, livestock or aquaculture industry.
3. El método para la producción de proteínas recombinantes, según reivindicación 1, CARACTERIZADO porque el gen de interés es clonado bajo el control de un promotor constitutivo como SV40, CMV, UBC, EFIA, PGK, CAG, MyHc o RSV; o bajo el control de un promotor específicos de la glándula mamaria como otSl- caseína, aS2-caseína, β-lactoglobulina, κ-caseína, β-caseína, promotores de la proteína acídica del suero y de a-lactoalbúmina. 3. The method for the production of recombinant proteins, according to claim 1, CHARACTERIZED because the gene of interest is cloned under the control of a constitutive promoter such as SV40, CMV, UBC, EFIA, PGK, CAG, MyHc or RSV; or under the control of a specific mammary gland promoter such as otSl-casein, aS2-casein, β-lactoglobulin, κ-casein, β-casein, serum acidic protein promoters and a-lactoalbumin.
4. El método para la producción de proteínas recombinantes, según reivindicación 1, CARACTERIZADO porque el vector AAV pueden derivarse de: 4. The method for the production of recombinant proteins, according to claim 1, CHARACTERIZED because the AAV vector can be derived from:
a. serotipos humanos 1, 2, 3, 4, 5, 6, 7, 8 o 9;  to. human serotypes 1, 2, 3, 4, 5, 6, 7, 8 or 9;
b. vectores quiméricos como los AAV-DJ o AAV-DJ/8; o  b. chimeric vectors such as AAV-DJ or AAV-DJ / 8; or
c. vectores cuyas cápsides ha sido modificada por cualquier herramienta combinatoria. C. vectors whose capsids have been modified by any combinatorial tool.
5. El método para la producción de proteínas recombinantes, según reivindicación 1, CARACTERIZADO porque el vector AAV contiene uno o más casetes de expresión. 5. The method for the production of recombinant proteins, according to claim 1, CHARACTERIZED in that the AAV vector contains one or more expression cassettes.
6. El método para la producción de proteínas recombinantes, según reivindicación 1, CARACTERIZADO porque el vector AAV puede ser producido mediante sistemas libres de ayudante basados en transfección transiente de un coctel de plásmidos, líneas celulares establemente productoras del vector AAV, o sistemas de producción en células de insecto, empleando baculovirus como vectores ayudantes.  6. The method for the production of recombinant proteins, according to claim 1, CHARACTERIZED because the AAV vector can be produced by helper-free systems based on transient transfection of a plasmid cocktail, stably producing cell lines of the AAV vector, or production systems in insect cells, using baculovirus as helper vectors.
7. El método para la producción de proteínas heterólogas, según reivindicación 1, CARACTERIZADO porque la solución que contiene los vectores AAV es una solución isosmótica que puede o no estar suplementada con un agente quelante. 7. The method for the production of heterologous proteins, according to claim 1, CHARACTERIZED because the solution containing AAV vectors is an isosmotic solution that may or may not be supplemented with a chelating agent.
8. Una solución isosmótica, según reivindicación 7, CARACTERIZADA porque el agente quelante se selecciona entre ácido l,2-bis(2-aminofenoxi)etano- N^N^N'-tetraacéticoC BAPTA), ácido dimercaptosuccínico (DMSA), ácido dietilentriamino pentaacético (DTPA), ácido 2,3-dimercapto-l-propanosulfónico (DMPS), ácido Nitrilo tri-Acético (NTA), ácido de etileno glicol tetraacético (EGTA), dimercaprol (INN) o, ácido etilen diamino tetraacético (EDTA). 8. An isosmotic solution according to claim 7, CHARACTERIZED in that the chelating agent is selected from l, 2-bis (2-aminophenoxy) ethane-N ^ N ^ N'-tetraacetic C BAPTA), dimercaptosuccinic acid (DMSA), diethylenetriamine acid pentaacetic acid (DTPA), 2,3-dimercapto-l-propanesulfonic acid (DMPS), tri-Acetic Nitrile acid (NTA), ethylene glycol tetraacetic acid (EGTA), dimercaprol (INN) or, ethylene diamine tetraacetic acid (EDTA) .
PCT/CL2015/000047 2014-09-12 2015-09-11 Method for producing recombinant proteins in the mammary glands of mammals by transforming the mammary gland epithelium with adeno-associated vectors WO2016037297A1 (en)

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