Patent Application
20140023711
The present invention is comprised within the field of molecular biology and biomedicine. Specifically, the present invention relates to a bioactive surface comprising: a polymer matrix, a connecting complex comprising at least one compound covalently bound to the surface of the polymer matrix, and a gene transfer vector bound to the connecting complex, where the compound of the complex is covalently bound to the transfer vector by means of a group selected from a carboxyl group, an amino group, an isocyanate group and a hydroxyl group, and to the use of said bioactive surface for transferring a nucleic acid to a cell. The present invention also relates to an implantable device characterized in that at least one part of its surface is coated with said bioactive surface and to the use of said implantable device for transferring a nucleic acid to a cell. The present invention also relates to a method for transferring a nucleic acid to a cell and to a kit for carrying out said method.
Gene transfer has a great therapeutic potential both in gene therapy and in tissue engineering, in addition to being very useful in research. The limiting factor in the development of gene transfer-based applications is the lack of effective systems for introducing the genetic material in cells. For gene transfer to be effective, nucleic acid must be transported to the target cells without it degrading, being taken into said cells, escape the intracellular degradation systems, being transported to the nucleus for transcription, and finally being translated into a protein. The use of viral or non-viral vectors also facilitates overcoming many intracellular barriers in the gene transfer process, the use of biomaterials allows solving problems associated with the transport of genetic material to target cells.
Substrate-dependent gene transfer, also called reverse transfection, is based on immobilizing viral or non-viral vectors on a surface which allows cell adhesion. This strategy allows reducing genetic material degradation and increasing the effective vector concentration, therefore a lower amount of nucleic acid is necessary, with the subsequent reduction in cytotoxicity. On the other hand, a tool which allows spatially regulating gene transfer process is obtained by combining the immobilization of the vector with a surface pattern.
The immobilization of the genetic material to be transferred can be non-specific or specific. Some biomaterials allow gene transfer vectors to be adsorbed directly on the surface; in this case the vectors interact with said biomaterials by means of non-specific mechanisms such as hydrophobic interactions, electrostatic interactions and van der Waals forces. Alternatively, specific interactions can be introduced by means of functional groups in the vector and the polymer, such as avidin-biotin or antibody-antigen.
Collagen and hyaluronic acid hydrogels having neutravidin on their surface are capable of binding DNA and biotinylated polyethylenimine cationic polymer complexes (Segura et. al. Biomaterials. 2005 May; 26(13): 1575-1584). The avidin-biotin system has also been used for immobilizing adenoviral vectors on chitosan supports (Hu et. al. J Gene Med. 2008 October; 10(10): 1102-1112). The immobilization of adenovirus on type I collagen gels modified with avidin using to that end a specific anti-adenovirus biotinylated antibody has also been described (Levy et. al. Gene Ther. 2001 May; 8(9):659-67). Likewise, adenoviruses have been immobilized by means of specific antibodies bound directly to polyurethane films (Stachelek et. al. Gene Ther. 2004 January; 11(1):15-24). It is important to highlight that specific immobilization strategy-based implantable devices have been used for in vivo gene transfer (Levy et. al. Gene Ther. 2001 May; 8(9):659-67; Klugherz et. al. Hum Gene Ther. 2002 Feb. 10; 13(3):443-54; Abrahams et. al; Stroke. 2002 May; 33(5):1376-82).
Regardless of the strategy used for immobilizing gene transfer vectors, vector immobilization and cellular uptake must be in equilibrium. The biomaterial used must be suitable for cell adhesion and also keep the gene transfer vector bound to the surface, but allowing cellular uptake of genetic material. If the bond is too weak the vectors will not be retained on the surface of the biomaterial for presentation to cells; in contrast, if vector immobilization is too strong, the uptake of genetic material may decrease significantly.
In conclusion, the enormous application potential of gene transfer in gene therapy and tissue engineering is significantly limited by the lack of effective, safe and controlled systems for gene transfer. The immobilization of genetic material in biomaterials is a strategy which allows improving efficacy and safety, as well as space-time control of the gene transfer process, provided that there is optimum equilibrium between the immobilization of genetic material and its cellular uptake.
The present invention relates to a bioactive surface, providing an effective, safe and controlled system for gene transfer. This bioactive surface of the invention comprises:
The present invention also relates to an implantable device characterized in that at least one part of its surface is coated with said bioactive surface and to the use of said implantable device for transferring a nucleic acid to a cell. Likewise, the present invention relates to a method for transferring a nucleic acid to a cell and to a kit for carrying out said method.
a) dishes coated with Amino-PEG. b) dishes coated with lentivirus.
The inventors used polystyrene matrices the surface of which had been previously modified by means of the covalent bond with pentafluorophenyl methacrylate (PFM). Treating the surface of the matrices with PFM proved effective for binding infective particles containing amino groups, particularly lentiviral and adenoviral particles, to said surface. These dishes treated with PFM and said particles are capable of genetically transducing human cells in a more effective manner than the same particles adsorbed in a non-specific manner on dishes which had not been treated with said molecule.
Therefore, a first aspect of the invention relates to a bioactive surface (hereinafter, bioactive surface of the invention) comprising:
As used herein, the term “bioactive surface” refers to any surface of a material with the capacity to transfer a biomolecule to a cell. Said biomolecule is preferably a nucleic acid. Therefore, in a preferred embodiment this term refers to any surface of a material having the capacity to genetically modify biological tissues or cells; the bioactive surface is therefore a gene transfer complex.
As used herein, “polymer matrix” is understood as any composite material that is biocompatible, biodegradable or non-biodegradable, formed by fibers, particles, or filaments embedded during the resinous or malleable phase. The polymer matrix of the bioactive surface of the invention is preferably selected from the list comprising: polystyrene, polycarbonate, poly(ethylene carbonate), polyethylene, polyolefins, poly(diol citrate), polyfumarate, polylactides, polycaprolactone, polyacrylamides, polysiloxanes, polyesters, polyamides, glycolic/lactic acid copolymers, polyurethane or any combination thereof. In a more preferred embodiment, the polymer matrix of the bioactive surface of the invention comprises polystyrene.
As used herein, the term “connecting complex” refers to a compound capable of binding the gene transfer vector to the polymer matrix by means of forming a covalent bond both with the polymer matrix and with the transfer vector. The connecting complex of the bioactive surface of the invention comprises at least one compound containing a group selected from a carboxyl group, an amino group, an isocyanate group and a hydroxyl group anchored to the surface of the polymer matrix.
The compound bound to the matrix preferably has the following formula (I)
where:
R1 is a radical which may or may not exist and if it exists, it is selected from a group comprising a (C1-C6) alkyl, a (C1-C6) alkenyl, a cycloalkyl or an aryl; preferably R1 does not exist,
R2 is a carboxyl group (—COO—), an amino group (—NH—), an ether group (—O—) or an isocyanate group (—CON—), preferably R2 is a carboxyl group,
R3 is a hydrogen, a (C1-C6) alkyl or a (C1-C6) alkene, preferably R3 is a hydrogen or a methyl group,
----- represents the binding of the compound of formula (I) to the gene transfer vector, and
represents the binding of the compound of formula (I) to the polymer matrix.
In the present invention, the term “alkyl” refers to radicals of linear or branched hydrocarbon chains having from 1 to 10 carbon atoms, preferably from 1 to 4, and binding to the rest of the molecule by means of a single bond, for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, tert-butyl, sec-butyl, n-pentyl, n-hexyl, etc. The alkyl groups can optionally be substituted with one or more substituents such as halogen, hydroxyl, alkoxyl, carboxyl, carbonyl, cyano, acyl, alkoxycarbonyl, amino, nitro, mercapto and alkylthio.
The term “alkenyl” refers to radicals of hydrocarbon chains containing one or more carbon-carbon double bonds, for example, vinyl, 1-propenyl, allyl, isoprenyl, 2-butenyl, 1,3-butadienyl, etc. Alkenyl radicals can optionally be substituted with one or more substituents such as halo, hydroxyl, alkoxyl, carboxyl, cyano, carbonyl, acyl, alkoxycarbonyl, amino, nitro, mercapto and alkylthio.
In the present invention, “cycloalkyl” refers to a stable 3 to 10-member monocyclic or bicyclic radical, which is saturated or partially saturated and which only consists of carbon and hydrogen atoms, such as cyclopentyl, cyclohexyl or adamantyl and which can optionally be substituted with one or more groups such as alkyl, halogen, hydroxyl, alkoxyl, carboxyl, cyano, carbonyl, acyl, alkoxycarbonyl, amino, nitro, mercapto and alkylthio.
In the present invention, the term “aryl” refers to a phenyl, naphthyl, indenyl, phenanthryl or anthracyl radical. The aryl radical can optionally be substituted with one or more substituents such as alkyl, haloalkyl, aminoalkyl, dialkylamino, hydroxyl, alkoxyl, phenyl, mercapto, halogen, nitro, cyano and alkoxycarbonyl.
The compound of formula (I) is preferably
where: ----- and have been described previously. This compound (1) preferably originates from PFM (pentafluorophenyl methacrylate).
In a preferred embodiment, the compound covalently bound to the polymer matrix is bound to the transfer vector by means of a carboxyl group. In a preferred embodiment, the compound covalently bound to the matrix is bound to the transfer vector by means of an amino group. In a preferred embodiment, the compound covalently bound to the polymer matrix is bound to the transfer vector by means of an isocyanate group. In a preferred embodiment, the compound covalently bound to the matrix is bound to the transfer vector by means of a hydroxyl group.
The connecting complex can further comprise a molecule selected from the list comprising: sugar, peptide, lipid or any combination thereof and where said molecule is located between the gene transfer vector and the carboxyl group, amino group, isocyanate group and hydroxyl group of the compound covalently bound to the polymer matrix, this molecule is preferably located between the R2 group of the compound of general formula (I) and the gene transfer vector. Therefore, the compound of general formula (I) and one of the molecules would comprise the connecting complex.
Therefore, in another preferred embodiment the connecting complex bound to the surface of the polymer matrix can bind to the transfer vector by means of a molecule selected from the list comprising: sugar, peptide, lipid or any of the combinations thereof.
The connecting complex of the bioactive surface of the invention can therefore comprise the compound containing a group selected from a carboxyl group, an amino group, an isocyanate group and a hydroxyl group anchored to the polymer matrix, preferably a PFM (pentafluorophenyl methacrylate) precursor, and at least one compound selected from the list comprising: a sugar, a peptide, a lipid or any combination thereof.
As used herein, the term “vector” or “gene transfer vector” refers to any system used for introducing an exogenous nucleic acid into a cell. The transfer vector must contain at least one reactive group through which it can react with the connecting complex, either with the compound anchored to the surface of the polymer matrix, or with another compound which can be part of the connecting complex, such as for example, but not limited to, a sugar, a peptide or a lipid. In a preferred embodiment, the gene transfer vector contains a reactive group through which it can react with the compound anchored to the polymer matrix, such as for example, but not limited to, an amino group or a carboxyl group. Therefore, in a preferred embodiment, the gene transfer vector of the bioactive surface of the invention comprises at least one amino group or one carboxyl group. For example, when the compound anchored, or covalently bound, to the surface of the polymer matrix is PFM, the gene transfer vector can react with the PFM if it contains an amino group. Therefore, in a more preferred embodiment the gene transfer vector of the bioactive surface of the invention comprises at least one amino group.
There are various viral and non-viral vectors that are known in the state of the art. Viral vectors suitable for putting the invention into practice include, but are not limited to the following: adenoviral vectors, adeno-associated vectors, retroviral vectors, lentiviral vectors, alpha viral vectors, herpes viral vectors, poxvirus-derived vectors and coronavirus-derived vectors. Non-viral vectors suitable for putting the invention into practice include, but are not limited to the following: polyamines, peptides, polypeptides, proteins, dendrimers, lipid/polyamine complexes and inorganic nanoparticles engineered for containing amino groups. The gene transfer vector of the bioactive surface of the invention can be a viral or non-viral vector. Therefore, in a preferred embodiment the gene transfer vector of the bioactive surface of the invention is selected from the list comprising: nucleic acid-polyamine complex, nucleic acid-peptide complex, nucleic acid-polypeptide complex, nucleic acid-protein complex, nucleic acid-dendrimer complex, nucleic acid-lipid-polyamine complex, nucleic acid-modified inorganic nanoparticle complex, adenoviral vector, adeno-associated vector, retroviral vector, lentiviral vector, alpha viral vector, herpes viral vector, poxvirus-derived vector or coronavirus-derived vector.
In a preferred embodiment, the gene transfer vector of the bioactive surface of the invention is a non-viral vector. In a more preferred embodiment, the non-viral gene transfer vector of the bioactive surface of the invention is a nucleoprotein complex, i.e., a complex formed by a nucleic acid and a peptide or polypeptide compound capable of facilitating the entry of the complex into the cell and of enhancing the biological function of the nucleic acid.
In another preferred embodiment, the gene transfer vector of the bioactive surface of the invention is a viral vector. In a more preferred embodiment, the viral gene transfer vector of the bioactive surface of the invention is a lentiviral vector, i.e., a lentiviral particle modified for reducing or eliminating its virulence, as well as for containing the nucleic acid to be transferred and for increasing the efficiency of said transfer. In another more preferred embodiment, the viral gene transfer vector of the bioactive surface of the invention is an adenoviral vector, i.e., an adenoviral particle modified for reducing or eliminating its virulence, as well as for containing the nucleic acid to be transferred and for increasing the efficiency of said transfer.
Therefore, in a preferred embodiment the bioactive surface of the invention comprises a polymer matrix and a gene transfer vector, preferably an adenoviral vector or a lentiviral vector, where said transfer vector is bound to the surface of said polymer matrix by means of a connecting complex comprising at least one PFM molecule bound to the surface of the polymer matrix of the invention.
The bioactive surface of the invention can be used either for completely or partially manufacturing an implantable device or for completely or partially coating an implantable device.
Therefore, a second aspect of the invention relates to an implantable device, hereinafter “implantable device of the invention”, characterized in that at least one part of its surface is coated with the bioactive surface of the invention.
As used herein, the term “implantable device” refers to a device which can be applied to the surface of the body of an animal, preferably a human, or which can be inserted into the body of the animal or of the human.
In a preferred embodiment, the implantable device of the invention is selected from the list comprising: a stent, an orthopedic prosthesis, a biocompatible membrane, a porous polymer, a patch, a suture, a capsule and a particle.
A third aspect of the invention relates to the use of the bioactive surface of the invention or of the implantable device of the invention for transferring a nucleic acid to a cell contacting the bioactive surface of the invention.
As used herein, the term “nucleic acid” refers to a polymeric form of nucleotides of any length, both ribonucleotides and deoxyribonucleotides. Therefore, the nucleic acid can be a deoxyribonucleic acid, such as for example, but not limited to, double-stranded DNA, single-stranded DNA or circular DNA, or a ribonucleic acid, such as for example, interference RNA. The nucleic acid is preferably DNA, more preferably double-stranded DNA, and yet more preferably double-stranded circular DNA.
The bioactive surface of the invention or the implantable device of the invention can be used for transferring a nucleic acid to a prokaryotic or eukaryotic cell. In a preferred embodiment, the bioactive surface of the invention or the implantable device of the invention is used for transferring a nucleic acid, preferably a DNA, to a eukaryotic cell. In a more preferred embodiment, the bioactive surface of the invention or the implantable device of the invention is used for transferring a nucleic acid, preferably a DNA, to an animal cell. In an even more preferred embodiment, the bioactive surface of the invention or the implantable device of the invention is used for transferring a nucleic acid, preferably a DNA, to a human cell.
A fourth aspect of the invention relates to a method for transferring a nucleic acid to a cell (hereinafter, “first method of the invention”) which comprises contacting the bioactive surface of the invention or the implantable device of the invention with said cell.
The first method of the invention can be used for transferring a nucleic acid to a prokaryotic or eukaryotic cell. In a preferred embodiment, the first method of the invention is a method for transferring a nucleic acid, preferably a DNA, to a eukaryotic cell. In a more preferred embodiment, the first method of the invention is a method for transferring a nucleic acid, preferably a DNA, to an animal cell. In an even more preferred embodiment, the first method of the invention is a method for transferring a nucleic acid, preferably a DNA, to a human cell.
A fifth aspect of the invention relates to a kit for carrying out the first method of the invention (hereinafter “first kit of the invention”) comprising:
In a preferred embodiment, the polymer matrix of the first kit of the invention is selected from the list comprising: polystyrene, polycarbonate, poly(ethylene carbonate), polyethylene, polyolefins, poly(diol citrate), polyfumarate, polylactides, polycaprolactone, polyacrylamides, polysiloxanes, polyesters, polyamides, glycolic/lactic acid copolymers, polyurethane or any combination thereof. In a more preferred embodiment, the polymer matrix of the first kit of the invention comprises polystyrene.
In a preferred embodiment, the connecting complex comprises a compound which is selected from the list comprising:
A sixth aspect of the invention relates to a kit for carrying out the first method of the invention (hereinafter “second kit of the invention”) comprising:
The elements which are necessary for forming a gene transfer vector depend on the type of gene transfer vector and are known in the state of the art.
In a preferred embodiment, the first or second kit of the invention comprises at least one element necessary for forming a gene transfer vector selected from the list comprising: polyamine, peptide, polypeptide, protein, dendrimer, lipid-polyamine, modified inorganic nanoparticle and plasmid encoding a capsid/envelope protein of a viral vector. Preferably, said viral vector is selected from the list comprising adenoviral vector, adeno-associated vector, retroviral vector, lentiviral vector, alpha viral vector, herpes viral vector, poxvirus-derived vector or coronavirus-derived vector.
In a preferred embodiment, the first or second kit of the invention comprises at least one element necessary for forming a nucleopeptide complex. Examples of elements necessary for forming a nucleopeptide complex include, but are not limited to, polylysine, fusogenic peptides, fusogenic proteins or lipid-peptide complexes.
In a preferred embodiment, the first or second kit of the invention comprises at least one element necessary for forming a lentiviral vector. Examples of elements necessary for forming a lentiviral vector include, but are not limited to, plasmids encoding capsid proteins of HIV, FIV, or EIAV; plasmids encoding viral envelopes having reduced toxicity and/or immunogenicity, and with tropism suitable for the cell type to be modified, such as for example, but not limited to, VSV-G, RD114, envelope proteins of LCMV, REV, filovirus, lysovirus, or rabies virus; plasmids encoding other lentiviral proteins, including those which confer lentiviral particles with a replicative and non-replicative nature, and also those which confer them with an integrative or non-integrative nature.
In a preferred embodiment, the first or second kit of the invention comprises at least one element necessary for forming an adenoviral vector. Examples of elements necessary for forming an adenoviral vector include for example, but are not limited to, plasmids encoding capsid proteins of type 2 or type 5 adenovirus. Said proteins could be modified for increasing or limiting their tropism or for reducing their immunogenicity and/or toxicity.
A seventh aspect of the invention relates to a method for preparing the bioactive surface of the invention (hereinafter, “second method of the invention”), which comprises contacting:
In a preferred embodiment, the second method of the invention comprises contacting the polymer matrix having the connecting complex bound to its surface with the gene transfer vector at a temperature between 20 and 40° C. for a time period between 5 minutes and 24 hours.
In a more preferred embodiment, the second method of the invention comprises contacting the polymer matrix having the connecting complex bound to its surface with the gene transfer vector at a temperature between 30 and 37° C. for a time period between 30 and 60 minutes.
In a more preferred embodiment, to favor the binding of the gene transfer vector to the polymer matrix having the connecting complex bound to its surface, the gene transfer vector is centrifuged on the polymer matrix having the connecting complex bound to its surface.
Said centrifugation is preferably performed at a speed between 100 and 20,000×g, and more preferably at a speed between 1,000 and 3,000×g. Said centrifugation is preferably performed at a temperature between 20 and 40° C. and for a time period between 5 minutes and 24 hours. More preferably, said centrifugation is performed at a temperature between 30 and 37° C. and for a time period between 30 and 60 minutes.
In another more preferred embodiment, to favor the binding of the gene transfer vector to the polymer matrix having the connecting complex bound to its surface, the matrix having the connecting complex bound to its surface is subjected to a negative pressure. Said pressure is preferably between −10−4 and −10−1 mBar, and more preferably between −9.10−3 and −9-10−2 mBar. Said negative pressure is preferably exerted at a temperature between 20 and 40° C. and for a time period between 5 minutes and 24 hours. More preferably, said negative pressure is exerted at a temperature between 30 and 37° C. and for a time period between 30 and 60 minutes.
In another more preferred embodiment, to favor the binding of the gene transfer vector to the polymer matrix having the connecting complex bound to its surface, the matrix is subjected to a combination of centrifugation and negative pressure. Said pressure is preferably between −10−4 and −10−1 mBar, and more preferably between −9.10−3 and −9.10−2 mBar. Said centrifugation is preferably performed at a speed between 1,000 and 20,000×g, and more preferably at a speed between 1,000 and 3,000×g. This combination is exerted at a temperature between 20 and 40° C. and for a time period between 5 minutes and 24 hours. More preferably, said combination is applied at a temperature between 30 and 37° C. and for a time period between 30 and 60 minutes.
In a preferred embodiment of the second method of the invention, the polymer matrix having the connecting complex bound to its surface is obtained by contacting said connecting complex with said polymer matrix under conditions which allow the polymer matrix to bind covalently to the compound of the connecting complex. In a preferred embodiment, the polymer matrix having the connecting complex bound to its surface is obtained by means of a method selected from the list comprising: chemical deposition, polymerization, plasma polymerization and iCVD (initiated chemical vapor deposition).
In a preferred embodiment of the second method of the invention, the polymer matrix having the connecting complex bound to its surface comprising at least one compound containing a group selected from a carboxyl group, an amino group, an isocyanate group and a hydroxyl group, preferably a PFM, is obtained by:
In another preferred embodiment, the connecting complex comprises a compound which is selected from the list comprising compounds (2) to (6) described above:
According to another preferred embodiment, the connecting complex further comprises a molecule selected from the list comprising sugar, peptide, lipid or any combination thereof, where said molecule can bind to the compound containing the connecting complex before or after it covalently binds to the polymer matrix.
Throughout the description and the claims, the word “comprises” and its variants do not intend to exclude other technical features, additives, components or steps. For the persons skilled in the art, other objects, advantages and features of the invention will be inferred in part from the description and in part from the practice of the invention. The following drawings and examples are provided by way of illustration and do not intend to limit the present invention.
The following specific examples which are provided in this patent document serve to illustrate the nature of the present invention. These examples are only included for illustration purposes and must not be interpreted as limiting the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting the field of application thereof.
Polystyrene was used as polymer matrix; specifically two types of easily available culture dishes were used: untreated polystyrene Petri dishes (PD) and cell culture (Tissue Culture, TC) dishes.
The surface of the dishes was treated with cold plasma for introducing pentafluorophenyl methacrylate (PFM) by means of grafting. The dishes treated with PFM were already prepared for reacting with a gene expression vector that had an amino group (
Given the protein nature of the infective particles (lentivirus, adenovirus, etc.), they have a high content of amino groups, so they can react with the treated surface.
For example, in the case of lentiviral particles (
Atomic force microscopy (AFM) was used to view the particles on the surface whereby the difference of the particles adhered to surfaces treated with PFM and to untreated surfaces can be seen. In the case of transduction efficiency, it was analyzed by means of introducing a reporter gene encoding green fluorescent protein (GFP) the expression of which can be detected by fluorescence microscopy and cytometry (cytometry also allowed quantifying transduction efficiency).
1.1 Coating the Polymer Matrix with PFM
To obtain polystyrene dishes chemically modified by means of PFM binding.
Polystyrene was used as polymer matrix; specifically two types of easily available culture dishes were used: untreated polystyrene Petri dishes (PD) and commercial cell culture (Tissue Culture, TC) dishes.
The dishes were exposed to cold Ar plasma for introducing the PFM by means of grafting. The method followed was the following:
To confirm that the dishes were well-coated with PFM, they were reacted with fluorescein isothiocyanate (FTSC) (which can be viewed by means of fluorescence microscopy). This was performed by means of a micro-contact printing assay consisting of using two buffers made of polydimethylsiloxane (PDMS) with the same logotype (IQS/CNM) that were previously submerged in a 0.5 mM FTSC solution for 1 hour. The buffers were gently deposited on the dishes, one on a dish treated with PFM and the other on a dish without PFM.
After washing with water for 30 minutes, the dishes were observed under a fluorescence microscope. The dishes treated with PFM clearly showed the IQS/CNM drawing formed by the FTSC molecules (
To view the infective particles on culture surfaces through AFM, to enable evaluating the efficiency of different treatments.
PD and TC dishes were used with the following treatments:
All the dishes were treated in a similar manner, varying the exposure time of the viral particles. The method was the following:
The following experiments were performed:
Experiment 1: TC Dishes with Lentivirus×30 Minutes
It can be seen in all cases that the surface with PFM contains what seems to be lentiviral particles given their size (150-200 nm). If a thorough observation is made, height up to 400 nm can be seen in some areas, which may indicate particle aggregation in these areas. In contrast, in the part without PFM, there are practically no particles greater than 30 nm.
Experiment 2: TC Dishes with Lentivirus×3 Hours
The results (
Experiment 3: PD Dishes with Lentivirus×3 Hours
In this case, a great difference is observed between one surface and another (
1.3. In Vitro Genetic Modification of Human Cells with Lentiviral Vectors
Experiment 1
This first experiment was intended for observing the difference between a TC dish and a TC dish treated with PFM. Furthermore, it was also intended for improving the diffusion of lentiviral particles so that they can react with the PFM by means of centrifugation.
The experiments performed were the following:
The materials used were:
The protocol used was the following:
The results show a great difference between the dishes treated with PFM and the untreated dishes (
Experiment 2
To evaluate the efficiency of gene transfer in dishes treated with PFM in comparison with surfaces of similar hydrophobicity with respect to those that are treated with PFM, but not reactive.
The following surfaces were used (
The experiments performed were the following:
The materials used were:
Before covering with the lentivirus, the dishes which will be used as negative controls were blocked. To that end, 1 ml of a 10 mM amino-PEG solution was added on a PD dish coated with PFM, and incubated overnight with orbital stirring. It was washed with milliQ water the next morning. To cover with the lentivirus, the same protocol described in Experiment 1 was followed, although in this case the cells were not analyzed by means of cytometry.
The obtained results (
Experiment 3
To demonstrate the selective genetic modification of human cells in vitro by means of contact with surfaces only partially treated with the method of the invention.
A TC dish was covered, half with PFM and the other half without PFM, and the experiment was conducted with the conventional protocol (viral suspension for 30 minutes at 37° C.)
To evaluate the efficacy of gene transfer in dishes treated with PFM by means of infection with adenovirus.
The materials used were:
The protocol used was the following:
The results (
To evaluate the efficacy of gene transfer in dishes treated with PFM by means of transfection with the commercial transfection agent, lipofectamine (Lipofectamine™, Invitrogen).
The materials used were:
The protocol used was the following:
The results (
To evaluate the efficacy of the transfer of siRNA to human cells by means of the surfaces of the invention, using RAWA-H8 peptide as a transfection agent (Barajas R. Development and Evaluation of RAWA-H8 Model as a Highly Efficient Peptide Transfection System [Desarrollo y evaluación del modelo RAWA-H8 como sistema peptidico de transfección de alta eficiencia], Doctoral Thesis, 2006).
The materials used were:
The protocol used was the following:
The results of the cytometry (
To evaluate the possibility of storing the surfaces of the present invention.
The surfaces used in this study were Petri dishes (PD) 30 mm in diameter treated or not treated with PFM.
To produce pluripotent cells (induced Pluripotent Stem, iPS) from mouse embryonic fibroblasts (MEF) by means of cellular reprogramming using the surfaces of the invention.
The dishes (PD, 30 mm) were modified with PFM according to the protocol described in preceding examples. The method of cellular reprogramming was the following:
The culture medium used was DMEM having a high glucose concentration (4.5 g/l) with glutamax and sodium pyruvate (GIBCO), 15% FBS, non-essential amino acids (1 mM), 0.1 mM beta-mercaptoethanol, 100 U/ml penicillin, 100 mg/ml streptomycin, 1,000 U/ml LIF (Leukemia Inhibitory Factor) and 2 mM valproic acid (valproic acid was only used on the first 7 days of culture).
As can be seen in
| Number | Date | Country | Kind |
|---|---|---|---|
| P 200931268 | Dec 2009 | ES | national |
This application is a national stage of International Application No. PCT/ES2010/070863 with the international filing date of Dec. 22, 2010 which claims the priority benefit of the Spanish Patent Application No. P200931268 filed on Dec. 24, 2009, the entire disclosures of which are incorporated herein by way of reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/ES10/70863 | 12/22/2010 | WO | 00 | 10/11/2013 |
