Classifications

• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • 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
  • C12N2799/00—Uses of viruses
  • C12N2799/02—Uses of viruses as vector
  • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid

Definitions

• the present invention relates to the construction of cloning vectors containing the max gene.
• the present invention relates to the introduction of the cloning vectors containing the max gene in cells, using transport vectors.
• the presence of cloning vectors containing the max gene in cells allow for the differential expression of the max gene in those cells.
• the present invention refers to a method of gene therapy in which the differential expression of the max gene has cytoprotective activity, especially neuroprotective activity, which may be applied to medical and to veterinary therapeutics for neurodegenerative conditions.
• Gene therapy is the set of techniques to insert and express exogenous genetic material into cells with therapeutic purposes.
• the transfer of genetic material can be done in vivo (directly into the target organism) or ex vivo through the transduction of cells that are then inserted into the target organism. That is, gene therapy can be combined with cell therapies, inclusive of stem cells.
• 'transport vector' a vehicle for insertion of the gene (known as 'transport vector'), to increase the probability of DNA entry.
• the most frequently used vectors in the current state of the art are derived from viruses, due to the fact that these microorganisms are naturally capable to infect living cells.
• 'non-viral' vectors such as plasmid DNA
• plasmid DNA a vector that introduce DNA in exogenous cells.
• non-viral vectors one can use microinjection, electroporation, plasmid injection, ballistic DNA injection, plasmids, liposomes, cationic compounds of lipid, protein or amidoprotein nature, such as DNA-calcium phosphate, DNA-DEAE dextran, DNA-lipid, DNA-lipid-protein and artificial chromosomes used in gene therapy.
• viral transport vectors one can use retrovirus, adenovirus, adeno- associated virus, hybrid adeno-adenoassociated virus, herpes simplex, lentivirus, foamy, HIV and vaccinia.
• the viruses are modified by recombinant DNA techniques for the removal of all genes associated with viral pathogenesis, in order to obtain a minimal construction capable of infecting cells without viral replication and without adverse effects.
• a gene of interest can be added to this construct, and the resulting construct is used to insert the gene of interest in cells of the organism to be treated. Nonetheless, the insertion and expression of a gene into an organism is an invasive process. Both the introduced gene as well as the viral vector may produce produce adverse effects related with the unbalance in gene expression, or the presence of non-self antigens.
• the target organism reacts to the presence of a virus by activatingg inflammatory and immune responses, which may damage the target of the gene therapy.
• One of the most competitive areas of biotechnology applied to gene therapy is the search for vectors which are both safer and, at the same time, capable of productive infection of the target cells, as well as of maintaining long term expression of the gene of interest.
• constructs derived from adeno-associated viruses have been targeted as a potential vectors for gene transfer in human clinical trials.
• AAV adeno-associated viruses
• their most favorable properties are: (I) no link of adeno- associated viruses with human diseases; (II) ability to infect cell lines derived from distinct tissues; (III) persistence of AAV-derived vectors in episomal form, thus avoiding insertional mutagenesis; (IV) ability to infect non-dividing cells.
• the capacity of transduction with recombinant AAV has been demonstrated in a wide variety of cell types, including differentiated cells, which suggests a great potential of this vector system for in vivo gene transfer to organs such as muscle, liver, central nervous sytem and lungs.
• the rAAV vectors are derived from plasmids that carry the inverted terminal repeats (ITRs) flanking the exogenous gene of interest.
• ITRs inverted terminal repeats
• the inverted terminal repeats are symmetrical sequences of 145 bases each, which are required for replication of the AAV DNA.
• rAAV vectors used to be produced by co-transfection of HEK293 cells with 2 plasmids, one containing the gene of interest flanked by ITRs, and a second plasmid containing the packing genes rep and cap, together with a helper virus (adenovirus or herpesvirus), the role of which was to provide ancillary factors for the replication of AAV.
• Modern techniques employ only the co-transfection of a main AAV plasmid containing the gene of interest and its regulatory sequences flanked by ITRs, plus a helper plasmid containing all the minimal sequences required for packaging.
• This procedure besides increasing production yield, aims at avoiding the contamination of rAAV preparation by adenovirus, which are pathogenic and, for this reason, were replaced by the minimal helper plasmid.
• the rAAV are recovered after cell lysis, and the helper plasmid is removed together with the DNA of the packaging cell line.
• three elements are required for packaging of the AAV: (I) eukaryotic cells in culture; (II) the main plasmid with the gene of interest and the replication sequence; (III) the helper plasmid with the viral replication genes.
• Recombinant AAV vectors are, in general, non-pathogenic and are the safest among the currently available vectors. Therefore, expression platforms based on rAAV have a great potentioal for clinical use.
• Another document, US 6,482,634, describes methods for the construction of vectors useful for the production of recombinant AAVs.
• the described method includes a host cell containing one molecule of 5 ' -3 ' nucleic acid, a P5 promoter from parvovirus, one spacer, one AAV rep sequence and one AAV cap sequence.
• the spacer reduces the expression of gene products rep78 and rep ⁇ .
• the second molecule of nucleic acid contains one minigene and this one, in turn, contains one transgene flanked by ITRs, which is under control of regulatory sequences directed to expression in the host cell and to essential functions for replication and packaging of rAAV.
• WO0182973 describes both viral and non-viral vectors as vehicles for the delivery of transgenes for the treatment of bone pathologies, through the local administration of a vehicle of delivery comprising the transgene for patients without a compatible bone matrix.
• This document describes a delivery viral or non-viral vector comprising genetic information related to therapeutic osteoinducer factors for target cells in vivo, forcing the cells to produce osteoinducer factors at the location of the bone pathology.
• the retina which is a part of the central nervous system, is subject to neurodegenerative diseases known as degenerative retinopathies or retinal dystrophies, which are current targets for the development of gene therapy.
• Examples of the combination of an adequate gene to a certain neurodegenerative pathology with a safe viral vector, in some cases already in clinical trials, can be found for the treatment of Parkinson ' s disease, Leber ' s congenital amaurosis and Alzheimer ' s disease.
• This document describes a form of therapeutical delivery of neurotrophins to a target (damaged, diseased or dysfunctional cholinergic neurons).
• the latter receive a transgene coding a neurotrophin within the mammalian brain, with the ensuing delivery of a defined concentration of recombinant neurotrophins by long periods.
• neuroprotective therapies that is, those that diminish in general the sensitivity of cells of the nervous sytem to PCD.
• Pharmacological methods based either on neurotrophic factors (a class of growth factors with action upon the nervous system) or on other neuroprotective molecules have been shown to be of little efficacy, because of the need of repetitive administration of the drugs.
• the solution to avoid the risk associated with repetitive manipulation of the cerebral tissue lies upon the development of therapies based on a reduced number of interventions, ideally a single one, with permanent results.
• Such an objective can be achieved through the development of gene therapy based on the discovery of target genes capable to protect the nervous tissue against multiple fators acting upon PCD.
• target genes capable to protect the nervous tissue against multiple fators acting upon PCD.
• identification of target genes for gene therapy of neurodegenerations is not trivial. Even in cases such as Huntington ' s disease, there is no single gene amenable to direct approach (which is usually the case in monogenic diseases).
• the procedure is specific for that condition, on the basis of selective sensitivity of a neuronal population to the presence of a certain neurotrophic factor, or to the expression of a certain gene for the functional recovery of a specific neuronal circuit within the central nervous system.
• the target of the gene therapy could possibly be the whole of the mechanisms of PCD.
• Several forms of execution of PCD have been identified, triggered as a consequence of multiple types of cellular stress.
• the best known form of execution is PCD by apoptosis.
• the experimental studies have clearly shown various pthways of apopotosis, as well as other alternative forms of PCD.
• the blockade of certain pathways of execution does not prevent cells from dying by alternative routes, which may be activated, for example, upon blockade of apoptosis itself [Guimaraes CA and Linden R - Programmed cell deaths. Apoptosis and alternative deathstyles. Eur J Biochem. 2004 May;271 (9): 1638-50].
• the retinal ganglion cells are the neurons whose axons form the optic nerve and are responsible for the transmission of the information processed within the retina to the upper levels of the visual system in the brain. Their degeneration results in blindness.
• transcription factor Max is excluded from the nucleus of the RGC early upon transection of their axons, and independent of the activity of caspases (proteases involved in neuronal death by apoptosis) [Petrs-Silva H, de Freitas FG, Linden R, Chiarini LB. Early nuclear exclusion of the transcription factor max is associated with retinal ganglion cell death independent of caspase activity [J Cell Physiol. 2004 Feb; 198(2): 179-87].
• Max codes for two isoforms of 21-22 kDa of the Max protein.
• Max functions in the control of the transcription of genes dependent of the activity of the transcription factor coded by the oncogene c-Myc.
• c-Myc must heterodimerize with Max.
• Max is capable of heterodimerization both with c-Myc, and with other proteins that comprise a network of transcription factors, such as Mnt and Mxd1-4 (previously known as Mad1 , Mxi1 , Mad3 and Mad4), as well as of homodimerization.
• Mnt and Mxd1-4 previously known as Mad1 , Mxi1 , Mad3 and Mad4
• the Myc-Max heterodimers and the Max-Max homodimers have affinity for the same elements in genomic DNA, and have opposed effects.
• the Max-Max homodimers antagonize the oncogenic effects of the Myc-Max heterodimers, and therefore, Max has an anti-oncogenic effect upon cells trans
• documents US 5,811 ,298 and US 6,140,476 describe plasmidial or viral vectors containing a chimeric gene composed by the coding sequence for a repressor domain of gene Mxi, fused to the sequence that codes for gene Max.
• the chimeric gene expressed by these vectors codes for a fusion protein called Rep-Max, which specifically blocks the tumor promoting activity of oncogenic proteins of the c-Myc family.
• cited documents do not overlap the present invention because: a) they refer to a fusion Mxi-Max construct, including only the bHLH, LZ and carboxy-terminal Max domains, that is, without the 5 ORF domain of gene max which codes for the the N-terminal Max domain that contains, for example, the phosphorylation sites of the Max protein, that is, a construction that is fundamentally distinct from the Max construction employed in the present invention; (b) relate to the repressor effect of the Mxi polypeptide domain, instead of the the effect of the polypeptide Max described in the present invention; (c) relate specifically to the repressor effect of the Mxi domain upon the tumor promoting activity of c-Myc family oncoproteins, distinct from the neuroprotective effect of Max described in the present invention; (d) relate to the effect of the Max poplypeptide exclusively as a facilitator of the repressor effect of the Mxi domain of the the fusion polypeptide, distinct from the direct effect of the Max polypeptide described in the present
• the described documents do not overlap the present invention because they relate to a chimera employing the max gene, and not the vector containing the max per se.
• the referred document does not cite the addition of the max gene to viral vectors with the aim of modulating the expression of the max gene with neuroprotective activity.
• the present invention relates to constructs of cloning vectors containing the max gene.
• the present invention refers to the introduction of cloning vectors containing the max gene in cell using transport vectors.
• the presence of the cloning vectors containing the max gene in cells allows the differential expression of the max gene with cytoprotective activity, especially neuroprotective activity, and may be applied to medical and veterinary therapeutics in neurodegenerative conditions.
• this invention consists of biological vectors containing the max gene, comprising: a) a cloning vector, comprising: a.1) a nucleotide sequence capable of expressing the max gene or its fragments; a.2) an adequate promoter; b) transport vectors, chosen among the group that comprises viral transport vectors, non-viral transport vectors, and mixtures of both.
• the cloning vector of a) comprises vectors of the group of plasmids, viruses, cosmids, and/or YACs.
• the promoter of b) is any sequence capable of directing the expression of the max gene and/or its fragments in a cell.
• the promotor is preferentially chosen among the group that comprises promoters CBA, CMV and/or hybrids CBA/CMV and/or specific promoters for each one of the various cell types.
• the promoter is chosen among the groups of neuron-specific promoters.
• the non-viral transport vectors are chosen among the group that comprises plasmids, liposomes, cationic lipid compounds,
• DNA-DEAE dextran, DNA-lipid, DNA-protein, DNA-lipid-protein, artificial chromosomes, nanoparticles, microinjection, eletroporation, injection of plasmid, ballistic injection of DNA.
• the viral transport vectors are chosen among the group comprising adenovirus, adeno-associated virus, recombinant adeno-associated virus, retrovirus, herpesvirus, lentivirus, foamy, HIV, vaccinia.
• An additional object of this invention consists of a method of production of biological vectors containing the max gene, comprising the stages of: a) providing a cloning vector containing: a.1) a nucleotide sequence capable of expressing the max gene or its fragments; a.2) an adequate promoter; b) providing a biological vector chosen among viral transport vectors, non-viral transport vectors, and mixtures of both; c) contacting the biological vector of b) with the cloning vector of a).
• An additional object of the present invention consists of a method of expression fo the max gene in cells, comprising the stage of contacting the biological vector with an adequate target cell, wherein the biological vector comprises: a) a cloning vector comprising: a.1) a nucleotide sequence capable of expressing the max gene or its fragments; a.2) an adequate promoter; b) transport vectors, chosen among the group that comprises viral transport vectors, non-viral transport vectors, and mixtures of both.
• the biological vector is present in a pharmaceutically acceptable vehicle, chosen among the group that comprises pharmaceutically acceptable excipients and carriers, convenient doses and treatments for use in particular compositions that can be described in a multitude of treatment regimens, including oral, parenteral, intravenous, intramuscular, intracerebral, intracerebroventricular and intraocular.
• a pharmaceutically acceptable vehicle chosen among the group that comprises pharmaceutically acceptable excipients and carriers, convenient doses and treatments for use in particular compositions that can be described in a multitude of treatment regimens, including oral, parenteral, intravenous, intramuscular, intracerebral, intracerebroventricular and intraocular.
• the cell is a neuron of a higher animal, for example, human being.
• An additional object of the present invention consists of a method of gene therapy comprising the stages of introduction of a biological vector containing the max gene in at least one target cell, where the differential expression of the max gene in the target cell will promote the modulation of cellular activity.
• the modulation of cellular activity has a cytoprotective function.
• the cell is a neuron of a higher animal, for example, human being. DESCRIPTION OF THE FIGURES
• Figure 1 shows a scheme of the transport vector pTR-CBA-max.
• Figure 2 shows a scheme of the transport vector pTR-SB-smCBA- max.
• Figure 3 shows a scheme of the transport vector pHpa-trs-SK- max.
• Figure 4 shows data on neuroprotection in vitro by rAAV-Max.
• Neonatal rats on the first postnatal day
• the animals were terminally anaesthesized, their eyes were removed and ther retinae were dissected.
• Explants of retinal tissue of approximately 1 mm 2 were cut and placed in culture medium with 5% fetal calf serum for 30 hours, after which the tissue was fixed with 4% paraformaldehyde.
• the rate of cell death in the retinal ganglion cell layer was estimated by the percentage of pyknotic nuclei, indicative of cell death by apoptosis,with respect to the total number of cells in the sampling area (means and standard errors of the means in the histogram).
• the explants of the retinae transduced with rAAV-Max had approximately 50% lower cell death rates than the explants transduced with the control rAAV-GFP.
• Increased expression of Max was confirmed by immunohistochemistry, whereas the expression of the GFP was confirmed by the intrinsic fluorescence of the control protein.
• Figure 5 shows data on neuroprotection in vivo by rAAV-Max. Rats were distributed among 4 experimental groups as shown by the scheme.
• fixation with 4% paraformaldehyde the eyes were removed and the whole retinae were flat-mounted on gelatinized glass slides for adhesion of the fixed tissue.
• Nuclei of live cells or pyknotic profiles indicative of cell death were stained with the DNA intercalator sytox green, which labels DNA with green fluorescence, and were quantified in the ganglion cell layer by counting in photomicrographs obtained under laser confocal microscopy.
• Counts were converted into nuclei (cell) densities and the results were presented in the form of means and standard errors of the means.
• the horizontal interrupted line indicates the mean density of displaced amacrine cells (in grey), found in the ganglion cell layer in the rat, which was discounted from the total to estimate the the density of ganglion cells (in black).
• the retinae transduced with rAAV-max on the first postnatal day were protected against the death of the ganglion cells.
• the retinae transduced on the same day as the optic nerve lesion did not show significant protection, due to the time required for the expression of the transgene carried by the pTR-CBA-max vector (approximately 14 days).
• Figure 6 shows data from another example of neuroprotection in vivo by rAAV-Max. Rats were distributed in 4 experimental groups as shown in the scheme. All animals received, on the first postnatal day, multiple injections into the superior colliculus (projection target of the RGC axons) of a suspension of red fluorescent microspheres, which are transported along the axons to the cell bodies of the RGC. This procedure serves to positively identify this type of neuron, which constitutes a model of neurodegeneration in the central nervous system. Three groups received injections of pTR-CBA-max in one eye, on either the 30th or the 60th postnatal day.
• 'cloning vectors' are vectors capable of amplifying the genetic information of a fragment of DNA inserted into an exogenous DNA and may be selected among a group that comprises plasmids, viruses, cosmids and/or YACs.
• the cloning vector contain a promoter, which is a sequence capable of promoting the expression of the DNA sequence in a cell.
• the promoter is preferentially chosen among a group that comprises CBA, CMV, and/or hybrids CBA/CMV, and/or specific promoters for target cells, such as for example, neuron-specific promoters.
• ' transport vectors' are the carriers of genetic information to target cells containing the cloning vector, and may be selected among a group that includes the viral and the non-viral transport vectors.
• 'viral transport vectors' are the group that comprises adenovirus, adeno-associated virus, retrovirus, herpesvirus, lentivirus, foamy, HIV, vaccinia.
• 'non-viral transport vectors' are those that comprise plasmids, liposomes, cationic lipid, protein and amidoprotein compounds, such as DNA-calcium phosphate, DNA-DEAE dextran, DNA-lipid,
• DNA-protein DNA-lipid-protein, artificial chromosomes, nanoparticles, microinjection, eletroporation, injection of plasmid, ballistic injection of DNA.
• 'biological vectors' are cloning vector(s) inserted in at least one transport vector.
• 'pharmaceutically accpetable' is a formulation containing acceptable pharmaceutical excipients and carriers, well known by professionals in the field, as also is the development of doses and treatments amenable to use in particular compositions that can be described in a multitude of treatment regimens, including oral, parenteral, intravenous, intranasal, intravitreous, intramuscular, intracerebral, intracerebroventricular and intraocular, as well as its administration and/or formulation.
• 'target cells' are the cells that will benefit from the expression of the max gene. Such cells include cells of higher animals, such as for example, neurons.
• 'modulation of activity' is any modification in the expression fo elements such as DNA, RNA and/or protein in the target cells, as well any change in the behavior of such cells during and/or after the expression of the max gene in the target cells, with the aim of promoting either the overexpression of the max gene or its silencing.
• the modulation shall be with the aim of promoting the overexpression of the max gene.
• the rAAV main plasmids are constructed based on plasmid pTR-
• CBA cken ⁇ -actin
• CMV cytomegalovirus
• the CBA-CMV combination produces great transduction efficiency in retinal cells, particularly in ganglion cells.
• the combination of promoter and enhancer can eventually, be modified for use in other cell types, especially other neuron types, within the scope of the present invention.
• pTR-SB-smCBA which does not contain the coding sequence of the gene of bacterial resistance to neomycine, its eukaryotic promoter HSV-tk, nor enhancer PYF441 from polyoma virus, nor the polyadenylation signal of the bovine growth hormone, which turns the vectors derived from this plasmid capable of use in clinical assays in human patients
• pHpa-trs-SK which is an rAAV vector contatining a DNA double strand, distinct from the above mentioned vectors.
• the base vector pHpa was given to our group by Dr.
• One pfu (1 ,5U/ 50 ⁇ l reaction) polymerase (Stratagene) was used for PCR (polimerase chain reaction) to generate the max clone flanked by the consensus sequence for cleavage by the Not ⁇ restriction enzyme.
• the primers used were forward (5'-gcggccgcatgagcgataacgat-3') and reverse (5 1 - gcggccgcttagctggcctccat-3').
• the generated clone was inserted into the TOPO bridge plasmid, following the protocol and reagents of the TOPO TA Cloning Kit (Invitrogen).
• the gene for GFP (green fluorescent protein) is routinely used as an experimental control, and had already been flanked by the Not ⁇ cleavage sequence in the pBIISK bridge plasmid.
• the plasmids containing inserts were digested with the Not ⁇ enzyme (Promega), in the concentration of 1U/ ⁇ g DNA for 1 hour at 37°C.
• the generated fragments were purified from agarose gels with the DNA cleaner kit (Invitrogen).
• the clones were ligated in the pTR-UF plasmid, also previously digested with Not ⁇ , dephosphorylated for 30 minute with SAP (shrimp alkaline phosphatase - Promega, 5U/ ⁇ g DNA) at 37 0 C, inactivated for 10 minutes at 75°C, purified with phenol-chloroform (Sigma), and precipitated with 3x volumes of 80% ethanol and 10% volume of 3M sodium acetate.
• the ligation reaction was done overnight with 1 ⁇ l of T4 DNA ligase (20,000U/ml - Promega) in a final volume of reaction of 20 ⁇ l, in an ice bath with temperature starting at 13°C and ending at 20 0 C.
• Colonies selected by the antibiotic ampicillin were grown in 5ml of LB medium with ampicillin for 14 hours in an incubator shaker at 200rpm at 37°C, to obtain enough plasmid for analysis.
• the bacteria in suspension were used to purify plasmids with the Wizard-plus mini-prep kit (Promega).
• the plasmids were analyzed for the presence and orientation of the clone, through DNA sequencing, using primers for the sequence of the pTR-UF plasmid: forward (5'-tcttttcctacagctcctgggcaa-3 J ) and reverse (5'- gcattctagttgtggtttgtccaaa-3').
• the plasmids were also digested with the Sma ⁇ restriction enzyme (Promega), at a concentration of 1 U/ ⁇ g DNA at room temperature for 1 hour, to confirm the presence of the clone, its orientation and that of the ITRs, which, being repetitive sequences, are easily excluded from the plasmid by bacteria in long-term cultures.
• Sma ⁇ restriction enzyme Promega
• helper plasmid contains only minimal sequences required for packaging and avoids the presence of adenovirus as a helper virus for the packaging process.
• Colonies of bacteria containing the constructs of interest were grown in suspension in 5ml of LB medium with ampicillin, in an incubator shaker at 200 rpm 37 0 C for 5 hours, then transferred to 1 L of TB medium (1 ,2% Triptone, 2,4% yeast extract and 0,4% glicerol), with 10% KHPO buffer plus 0,1 % ampicillin (100mg/ml), pre-heated at 37°C.
• the colonies in LB medium were left in the shaker at 37 0 C for another 14h.
• the medium was centrifuged for 10 minutes at 4000 rpm, using a JA-10 rotor.
• RNAse For each 250ml of centrifuged medium, 20ml of resuspension solution (5OmM Tris- HCI, pH8,0, 1 OmM EDTA, pH 8,0, 20 ⁇ g/ml RNAse) were added to the pellet, and the latter was dissociated with a pipet. Then, an additional 20ml of a previously prepared lysis solution were added (0,2N NaOH, SDS 1% (w/v)). The mixture was shaken and incubated for 5 minutes at room temperature. Lastly, 20 ml of neutralization solution (3M potassium acetate, 28,7% acetic acid (w/v)) were added under shaking.
• neutralization solution 3M potassium acetate, 28,7% acetic acid (w/v)
• the pellet When dry, the pellet was carefully disolved in 10 ml TE (10OmM Tris-HCI pH 7,6, 1OmM EDTA pH 8,0) with a pipette. Then, 1g cesium chloride was added for each 1 ml of solution. The mixture was transferred to ultracentrifuge tubes (25,4 X 89,1 mm, Quick-Seal - Beckman), and 100 ⁇ l ethidium bromide were added (740 ⁇ g/ml). (Invitrogen). The tubes were sealed and centrifuged overnight, at 45,000 rpm, using a Vti-65 rotor.
• DNA concentration was measured by UV (ultra-violet light) absorption, using a spectrophotometer. Five ⁇ l of DNA were added to 995 ⁇ l H 2 O, and optical density was read at 260nm excitation. To analyze the purity of the sample, a second measurement was made at 280nm excitation. The ratio between the two readings at 260nm and 280nm was determined. When the ratio is between 2 and 1.5 the plasmids can be used for transfection. Below 1.5 there is the need for further purification.
• HEK-293 human embryonic kidney cell line
• DMEM Dulbecco ' s modified Eagle ' s medium
• FBS fetal bovine serum
• Gibco penicillin G
• Transfection was done by precipitation with calcium phosphate.
• a mixture was made of 1 ,8 mg of the helper plasmid pDG plus 0,6 mg of the main plasmid, such as to yield a 1 :1 molar ratio of DNA, in 50ml de CaCI 2 0,25M, followed by addition of 50ml 2X HBS, pH 7,05 (25OmM HEPES free acid, 1.4M NaCI and 14mM Na 2 HPO 4 ).
• the 2X HBS solution is added last.
• the mixture was incubated for 1-2 minutes at room temperature and then added to 1100ml complete medium.
• the cells ' culture medium was removed and the new medium was added. The cells were incubated for 72 hours.
• lodixanol 5, 5'-[(2-hydroxy-1 ,3- propanediyl)bis(acetyl-1 ,3 ⁇ benzenecarboxamide] (OptiPrep - Axis-Shield)
• lodixanol is a non-ionic medium for the formation of density gradients, which remains isosmotic at all densities, and increases the efficacy of purification of viral vectors, avoiding both the loss of infectivity and the toxicity of cesium chloride or sucrose (Zolotukhin, S.
• rAAV The density of rAAV is about 1 ,266g/ml, which is equivalent to 50% iodixanol. Therefore, a discontinuous gradient is generated in tubes (28,8 X 107,7 mm, Quick-seal - Beckman), by laying the 15ml of the least dense cell lysate, followed by 5ml 15% iodixanol (Sigma) in PBS-MK (PBS with 1 mM MgCI 2 and 2,5mM KCI), 15ml 25% iodixanol, 15ml 40% iodixanol, and finally 15ml 60% iodixanol.
• PBS-MK PBS with 1 mM MgCI 2 and 2,5mM KCI
• the fraction obtain through the iodixanol gradient was then purified and concentrated by column chromatography.
• a 5 ml heparin HiTrap ionic exchange column (Pharmacia) was used in the FPLC AKTA system (Pharmacia), at 5ml/minute and eluted with PBS-MK with 0,5M NaCI.
• the column was initially equilibrated at 5ml/min with 25ml buffer A (2OmM Tris-HCI, 15mM NaCI, pH 8,5), then with 25ml buffer B (2OmM Tris-HCI, 50OmM NaCI, pH 8,5), followed by 25ml buffer A.
• the iodixanol vector fraction was diluted 1 :1 in buffer A, and applied to the column, which was adjusted to a flow rate of 3-5 ml/min. After the sample had flown through the HiTrap column, it was washed with 50ml buffer A. The vector was eluted in buffer B, fractioned in approximately 50 fractions of 1 ml, and the fractions containing vector were collected. The vector was then concetrated and desalinized in a Biomax 100K concentrate (Millipore) for 3 cycles of centrifugation in microfuge at maximum 14000 rpm speed for 1 minute. In each cycle, the virus was concentrated to 1 ml, followed by the addition of 10ml de Ringer Lactate. The rAAV vectors were stocked at -80 0 C.
• Example 8.1 - EXTRACTION OF PROTEINS Fifteen ⁇ l of each stock were mixed to sample buffer (45% glycerol 100%, 5% 2- ⁇ -mercapto-ethanol, 2% SDS in Tris- HCI 0,5 M pH 6,8, and Bromophenol Blue), boiled for 5 minutes and applied to a 12% de polyacrilamide gel (Bio-Rad).
• Example 8.2 - SILVER STAINING The vector stocks were examined for purity by analysis of the total proteins in polyacrilamide gels stained with silver, using a Bio-Rad kit.
• EXAMPLE 9 - TITRATION The stock of purified vector was treated with DNAse I to digest any unpackaged contaminant DNA. Ten ⁇ l of the purified vector stock were incubated with 10U DNAse I (Boehringer) in 100 ⁇ l of reaction mixture (5OmM Tris-HCI, pH 7,5, 1OmM MgCI 2 ) for 1 hour at 37°C.
• 10 ⁇ l of 10X proteinase K buffer (1OmM Tris-HCI, pH 8,0, 1OmM EDTA, 1% SDS) was added, followed by addition of 1 ⁇ l Proteinase K (18,6mg/ml, Boehringer).
• the mixture was incubated at 42°C for 1 hour.
• the viral DNA was purified in 2 extractions with phenol/chloroform, followed by one extraction with chloroform and precipitation with ethanol overnight at -20 0 C, using 10 ⁇ g glycogen as a carrier.
• the mixture was then centrifuged in a microfuge for 30 minutes, at maximum 14000 rpm speed.
• the precipitated DNA was resuspended in 100 ⁇ l water.
• Each reaction mixture for PCR had 1 ⁇ l viral DNA diluted in 2 series of dilutions of the standard plasmid, which can be the main plasmid used to produce the viral vector, with known concentration, but with a distinct gene from the vector-contained transgene.
• the best rates for this standard lie between 1 and 100pg, and other dilutions used are equivalent to expression rates of 1 , 10, 50 e 100pg.
• Primers used refer to the sequence immediately up and downstream od the transgene insertion: forward (5'- agttattaatagtaatcaatta-3') e reverse (5'-atcctttcagggtattccagta-3').
• the products of each reaction were analyzed in 2% agarose (Invitrogen) gel, containing 0,5 ⁇ g/ml ethidium bromide.
• the electrophoresis ran until the 2 bands were resolved such as to allow comparison. Images of the bands were acquired using an ImageStore 7500 UVP system (BioRad). The density of each band was measured using an image analysis software (ZERO-Dscan Image Analysis System, versioni .O - Scanalytics). Rates were plotted as a function of the concentration fo the standard DNA.
• the concentration of the viral vector stock corresponded to equal numbers of molecules of viral DNA and standard DNA.
• EXAMPLE 10 - VETOR PTR-CBA-MAX (FIGURE 1): Constructed upon a serotype 2 rAAV base vector (pTR), it contains a neomycin resistance gene for selection and a CBA (chicken beta-actin) general promoter, capable of promoting the expression of max in any cell type. Vectrors derived from AAV show a relatively slow time course of expression, and reach maximum expression of the transgene about 14 days following transfection. The pTR-CBA-max vector reduced the sensitivity of retinal ganglion cells to cell death after transection of their axons in both in vitro and in vivo experiments.
• Neonatal rats (on the first postnatal day) received injections of hte pTR-CBA-max in one eye, After 14 days, the animals were terminally anaesthesized, their eyes were removed and fixed by immersion in 4% paraformaldehyde in phosphate buffer. The eyes were cut in a cryostat and sections 10 micrometers thick were monted in glass slides for immunohistochemistry.
• the Max protein was detected by reaction with a specific antibody, followed by development with a red fluorescent secondary antibody.
• the protein class III beta-tubulin, which labels RGC, was detected with a specific antibody, followed by development with a green fluorescent secondary antibody. Photomicrographs were obtained through laser confocal microscopy.
• NEUROPROTECTION IN VITRO BY RAAV-MAX Neonatal rats (on the first postnatal day) received injections of pTR-CBA-Max in one eye and pTR-CBA-GFP (control) on the other eye. After 14 days, the animals were terminally anaesthesized, their eyes were removed and the retinae were dissected. Retinal tissue explants of approximately 1mm 2 were cut and maintained in culture medium with 5% fetal calf serum for 30 hours, after which the tissue was fixed in 4% paraformaldehyde.
• the rate of cell death in the ganglion cell layer was estimated by the percentage of pyknotic profiles, indicative of cell death by apoptosis, with respect to the the total number of cells in the sample (means and standard errors of the means in the histogram).
• the retinal transduced with rAAV-max had approximately 50% lower rates of cell death than the explants transduced with the control rAAV-GFP ( Figure 4).
• the increased expression of Max was confirmed by immunohistochemistry, while the expression of GFP was confirmed by the intrinsic fluorescence of the latter protein (Petrs-Silva et al, unpublished results)
• Rats were distributed among 4 experimental groups as shown by the scheme of Fig. 5. Two groups received injections of pTR-CBA-max in one eye, either on the first or on the 60th postnatal day. Three groups received lesions of the optic nerve on the 60th postnatal day (on the injected side, when injected) and all 4 groups were terminally anaesthesized and killed on the 74th postnatal day. Following fixation with 4% paraformaldehyde, the eyes were removed and the whole retinae were flat-mounted on gelatinized glass slides for adhesion of the fixed tissue.
• Nuclei of live cells and pyknotic profiles indicative of cell death were stained with the DNA intercalator sytox green, which labels DNA with green fluorescence, and were quantified in the ganglion cell layer by counting in photomicrographs obtained by laser confocal microscopy. Counts were converted into nuclei (cell) densities and the results were presented in the form of means and standard errors of the means.
• the horizontal interrupted line indicates the mean density of displaced amacrine cells (in grey), found in the ganglion cell layer in the rat, which was discounted from the total to estimate the the density of ganglion cells (in black).
• the retinae transduced with rAAV-max on the first postnatal day were protected against the death of the ganglion cells.
• NEUROPROTECTION IN VIVO BY RAAV-MAX Rats were distributed in 4 experimental groups as shown in the scheme of Fig. 6. All animals received, on the first postnatal day, multiple injections into the superior colliculus (projection targets of the RGC axons) of a suspension of red fluorescent microspheres, which are transported along the axons to the cell bodies of the RGC.
• This procedure serves to positively identify this type of neuron, a models of neurodegeneration in the central nervous system.
• Three groups received injections of pTR-CBA-max in one eye, on either the 30th or the 60th postnatal day.
• Three groups received optic nerve lesions by crush, on the 60th day, on the injected side, when injected), and all 4 groups were terminally anaesthesized and killed on the 74th postnatal day.
• fixation with 4% paraformaldehyde the eyes were removed and the whole retinae were dissected and prepared as flat mounts on gelatinized glass slides for adhesion of the fixed tissue.
• the RGC were identified by the red labeling with the microspheres, whereas all nuclei of cells in the ganglion cell layer were labeled with the DNA intercalant sytox green (green).
• the RGC were quantified by counts in photomicrographs obtained by confocal microscopy.. The counts were transformed in nuclei (cell) densities and the results were shown in the form of means and standard errors of the means.
• EXAMPLE 11 - VECTOR PTR-SB-SMCBA-MAX (FIGURE 2): Under construction upon the serotype 2 rAAV base vector (pTR), similar to the base vector used in design 1. This vector had deleted the sequences coding for the neomycin bacterial resistance gene, its eukaryotic promoter HSV-tk, the PYF441 polyoma virus enhacer, and the bovine growth hormone polyadenylation signal. These modifications fo not alter the speed of transgene expression, nor the distribution of the vector in the nervous system. The changes, however, make the vector amenable for use in human clinical assays.
• EXAMPLE 12 - VECTOR PHPA-TRS-SK-MAX (FIGURE 3): Under construction upon a serotype 2 scAAV base vector (pHpa), distinct from the base vectors described above.
• the pHpa base vector was modified by the group of Dr. William Hauswirth, at the University of Florida, such as to accelerate the expression of the transgene due to the formation of a double strand, thus skipping the relatively slow stage of synthesis of the complementary strand.
• the promoter is CMV (cytomegalovirus).
• This type of vector expresses the transgene in up to 7 days, that is, reducing by 50% the time needed for transgene expression. Tests done in our laboratory confirmed the acceleration of the expression of the GFP transgene transduced by this vector in retinal tissue (Petrs-Silva et al, unpublished results).

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