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WO2016037297A1 - Méthode pour la production de protéines de recombinaison dans la glande mammaire de mammifères par la transformation de l'épithélium glandulaire mammaire avec des vecteurs adénoassociés - Google Patents

Méthode pour la production de protéines de recombinaison dans la glande mammaire de mammifères par la transformation de l'épithélium glandulaire mammaire avec des vecteurs adénoassociés 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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aav
production
recombinant proteins
milk
interest
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PCT/CL2015/000047
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English (en)
Spanish (es)
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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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    • 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

La présente invention concerne une méthode qui permet de produire des protéines de recombinaison dans le lait de mammifères non transgéniques. Le procédé repose sur le transfert direct de l'information génétique présentant un intérêt aux cellules qui composent l'épithélium de sécrétion mammaire, au moyen de l'utilisation de vecteurs adénoassociés. Au moyen de cette méthode il est possible de produire de hauts niveaux de protéines de recombinaison dans le lait de mammifères non transgéniques pendant une durée de lactation complète. Cette méthode est modulable et permet d'atteindre des niveaux soutenus d'expression sur des périodes supérieures à 100 jours.
PCT/CL2015/000047 2014-09-12 2015-09-11 Méthode pour la production de protéines de recombinaison dans la glande mammaire de mammifères par la transformation de l'épithélium glandulaire mammaire avec des vecteurs adénoassociés WO2016037297A1 (fr)

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CL2014002410A CL2014002410A1 (es) 2014-09-12 2014-09-12 Método para la producción de proteínas recombinantes en glándula mamaria de mamíferos mediante la transformación del epítelio glandular mamario con vectores adenoasociados.

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WO2020206566A1 (fr) * 2019-04-11 2020-10-15 Centro De Biotecnología Y Biomedicina Spa Anticorps monoclonal recombinant contre le vegf exprimé dans la glande mammaire d'un animal non transgénique et procédé d'obtention de celui-ci

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