EP1513559A1 - Increased delivery of a nucleic acid constrtuct in vivo by the poly-l-glutamate (plg) system - Google Patents
Increased delivery of a nucleic acid constrtuct in vivo by the poly-l-glutamate (plg) systemInfo
- Publication number
- EP1513559A1 EP1513559A1 EP03741818A EP03741818A EP1513559A1 EP 1513559 A1 EP1513559 A1 EP 1513559A1 EP 03741818 A EP03741818 A EP 03741818A EP 03741818 A EP03741818 A EP 03741818A EP 1513559 A1 EP1513559 A1 EP 1513559A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- seqid
- nucleic acid
- acid expression
- expression construct
- polypeptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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Classifications
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- 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
- A61K48/0008—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 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
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- 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
Definitions
- nucleic acid expression construct having a specific encoded gene i.e. a plasmid
- plasmid a nucleic acid expression construct having a specific encoded gene
- both methods of protein supplementation work well, there are a number of advantages to the nucleic acid expression construct supplementation method when compared to the administration of recombinant proteins, for example: the conservation of native protein structure; improved biological activity; avoidance of systemic toxicities; and avoidance of infectious and toxic impurities.
- the plasmid mediated gene supplementation method allows the subject to have prolonged exposure to a therapeutic range of the therapeutic protein, as demonstrated by the persistent levels of the therapeutic protein found in the subjects circulation system.
- injections of the plasmid constructs can promote the production of enzymes and hormones in subjects in a manner that more closely mimics the natural process. Furthermore, among the non-viral techniques for gene product supplementation in vivo, the direct injection of plasmid DNA into muscle tissue is simple, inexpensive, and safe.
- a plasmid based expression system can be composed of a synthetic gene delivery system in addition to the nucleic acid encoding a therapeutic gene products. In this way many of the risks associated with viral vectors can be avoided.
- the plasmid (i.e. a non-viral expression system) products generally have low toxicity due to the use of "species-specific" components for gene delivery, which minimizes the risks of immunogenicity generally associated with viral vectors.
- plasmids As episomal systems residing outside the chromosomes, plasmids have defined pharmacokinetics and elimination profiles, leading to a finite duration of gene expression in target tissues (Houk et al., 2001; Mahato et al., 1997).
- the delivery of plasmid DNA into a cell by electroporation involves the application of a pulsed voltage electric field to create transient pores in the cellular membrane that allows for the influx of exogenous plasmid DNA molecules (Smith and Nordstrom, 2000).
- a pulsed voltage electric field By adjusting the electrical pulse generated by an electroporetic system, the efficiency of nucleic acid molecules that travel through passageways or pores can be regulated.
- U.S. Patent 5,704,908 describes an electroporation apparatus for delivering molecules to cells at a selected location within a cavity in the body of a patient.
- These pulse voltage injection devices are also described in U.S. Patent Nos. 5,439,440 and 5,702,304, andPCT WO 96/12520, 96/12006, 95/19805, and 97/07826.
- Electroporation technique has been used previously to transfect tumor cells after injection of plasmid DNA (Nishi et al., 1997; Rols et al., 1998), or to deliver the antitumoral drug bleomycin to cutaneous and subcutaneous tumors (Belehradek et al., 1994; Heller et al., 1996). Electroporation also has been used in rodents and other small animals, e.g. (Muramatsu et al., 1998; Aihara and Miyazaki, 1998; Hasegawa et al., 1998; Rizzuto et al., 1999).
- Electroporation is the use of an electric field to induce transient permeabilization of bio-membrane pores, and allows macromolecules, ions, and water to pass from one side of the membrane to the other. Thus, electroporation has been used to introduce drugs, DNA or other molecules into multi-cellular tissues.
- the technique has been used in vivo initially to transfect tumor cells after injection of plasmid DNA (Rols et al., 1998), or to deliver the antitumoral drug bleomycin to cutaneous and subcutaneous tumors (Allegretti andPanje, 2001; Heller et al., 1996). Recently, numerous studies, mostly on small mammals, showed that the technique increases dramatically plasmid uptake by skeletal muscle cells, and allows production of peptides at therapeutic levels (Yasui et al., 2001; Yin and Tang, 2001).
- GHRH human growth hormone releasing hormone
- transfection facilitation polypeptide e.g. polyL-glutamate (“PLG)
- PLG polyL-glutamate
- the transfection facilitating polypeptide may bind to surface of proteins and facilitate the uptake by increasing the bio-availability, neutralizing the normal degradation process in the interstitial fluid (i.e. protecting the DNA from the nucleases present in the interstitial fluid).
- a transfection facilitating polypeptide may prevent transport of DNA into the lysosomes (i.e. organelles where foreign DNA and / or proteins are degraded in the cells) by disruption of microtubule assembly (Fujii etal., 1986).
- transfection facilitating polypeptides e.g. PLG groups
- transfection facilitating polypeptides have been used to increase stability of anti-cancer drugs (Li et al., 2000), and as "glue” to close wounds or to prevent bleeding from tissues during wound and tissue repair (Otani et al., 1998; Otani et al., 1996).
- transfection facilitating polypeptides do not enhance an immune response or the production of antibodies. It should be emphasized that some evidence suggests that certain transfection facilitating polypeptides may only effective in conjunction with the method of electroporation. Furthermore, PLG has been demonstrated to decrease muscle damage associated to plasmid delivery (Draghia-Akli et al., 2002a).
- One aspect of the current invention is a composition for facilitating the electrophoretic transfer of a nucleic acid expression construct into the cells of a recipient, wherein the nucleic acid expression construct can express an encoded gene in the recipient.
- the composition of the invention comprises a nucleic acid expression construct that is associated with a charged transfection-facilitating polypeptide.
- the composition is prepared wherein a ratio in moles of the charged transfection-facilitating polypeptide to nucleic acid expression construct comprises from 1 mole to 5,000 moles of the charged transfection- facilitating polypeptide per mole of nucleic acid expression construct.
- the ratio in moles is equal to 1 mole of the nucleic acid expression construct to 1,200 moles or less of the charged transfection-facilitating polypeptide, and in another preferred embodiment, the ratio in moles is equal to 1 mole of the nucleic acid expression construct to 1 mole of the charged transfection-facilitating polypeptide.
- the transfection-facilitating polypeptide comprises a charged polypeptide (e.g. poly-L-glutamate).
- the nucleic acid expression construct comprises SeqID#l 1, SeqID#12, SeqID#13, SeqID#14, SeqID#17, SeqID#18, SeqID#19, SeqID#20, or SeqID#21.
- the nucleic acid expression construct encodes a growth-hormone-releasing- hormone ("GHRH") or functional biological equivalent thereof, as embodied in HV-GHRH (SEQID# 1), TI-GHRH (SEQID# 2), TV-GHRH (SEQID# 3), 15/27/28-GHRH (SEQID# 4), wt-GHRH (SEQID#5).
- GHRH growth-hormone-releasing- hormone
- a second aspect of the current invention is a method for introducing a nucleic acid expression construct into a cell of a selected tissue in a recipient.
- the method comprises penetrating the selected tissue with a plurality of needle electrodes, wherein the plurality of needle electrodes are arranged in a spaced relationship, introducing a composition comprising nucleic acid expression construct having an associated charged transfection- facilitation polypeptide, and applying an electrical pulse to the plurality of needle electrodes.
- caliper electrodes can also be used an alternative to needle electrodes.
- composition is prepared wherein a ratio in moles of the charged transfection-facilitating polypeptide to nucleic acid expression construct comprises from 1 mole to 5,000 moles of the charged transfection-facilitating polypeptide per mole of nucleic acid expression construct, hi a preferred embodiment, the ratio in moles is equal to 1 mole of the nucleic acid expression construct to 1,200 moles or less of the charged transfection-facilitating polypeptide, and in another preferred embodiment, the ratio in moles is equal to 1 mole of the nucleic acid expression construct to 1 mole of the charged transfection-facilitating polypeptide.
- the transfection-facilitating polypeptide comprises a charged polypeptide (e.g. poly-L-glutamate).
- the nucleic acid expression construct comprises SeqID#l l, SeqID#12, SeqID#13, SeqID#14, SeqID#17, SeqID#18, SeqID#19, SeqID#20, or SeqID#21. Additionally, the nucleic acid expression construct encodes a growth-hormone-releasing-hormone ("GHRH”) or functional biological equivalent thereof, as embodied in HV-GHRH (SEQID# 1), TI-GHRH (SEQID# 2), TV-GHRH (SEQID# 3), 15/27/28-GHRH (SEQID# 4), wt-GHRH (SEQID#5).
- GHRH growth-hormone-releasing-hormone
- a third aspect of the current invention is a method to increase stability of a nucleic acid expression construct, comprising: mixing the nucleic acid expression construct with a charged transfection-facilitating polypeptide, wherein charged transfection-facilitating polypeptide comprises a poly-L-glutamate polypeptide and the nucleic acid expression construct is utilized for plasmid mediated gene supplementation.
- the method involves making a composition that is prepared wherein a ratio in moles of the charged transfection- facilitating polypeptide to nucleic acid expression construct comprises from 1 mole to 5,000 moles of the charged transfection-facilitating polypeptide per mole of nucleic acid expression construct.
- the ratio in moles is equal to 1 mole of the nucleic acid expression construct to 1,200 moles or less of the charged transfection-facilitating polypeptide, and in another preferred embodiment, the ratio in moles is equal to 1 mole of the nucleic acid expression construct to 1 mole of the charged transfection-facilitating polypeptide.
- the transfection-facilitating polypeptide comprises a charged polypeptide (e.g. poly-L-glutamate).
- the nucleic acid expression construct comprises SeqID#l l, SeqID#12, SeqID#13, SeqID#14, SeqID#17, SeqID#18, SeqID#19, SeqID#20, or SeqID#21.
- the nucleic acid expression construct encodes a growth-hormone-releasing-hormone ("GHRH") or functional biological equivalent thereof, as embodied in HV-GHRH (SEQID# 1), TI-GHRH (SEQID# 2), TV-GHRH (SEQID# 3), 15/27/28-GHRH (SEQID# 4), wt-GHRH (SEQID#5).
- GHRH growth-hormone-releasing-hormone
- Figure 1 shows an electrode array of the prior art using six electrodes in three opposed pairs. It further depicts a single centralized electroporation overlap point, which is the center point of the asterisk pattern illustrated;
- Figure 2 shows one electrode array of the present invention using five electrodes. It further depicts how a symmetrically arranged needle electrode array without opposing pairs can produce a decentralized pattern during an electroporation event in an area where no congruent electroporation overlap points develop and how an area of the decentralized pattern resembles a pentagon;
- Figure 3 shows a the serum levels of SEAP in mice that were injected with an expression plasmid pSP SEAP coated with various concentrations of poly-L-glutamate;
- Figure 4 shows a the serum levels of SEAP in pigs that were injected with an expression plasmid pSP SEAP coated with and without poly-L-glutamate.
- Figure 5 shows a the serum levels of SEAP in dogs that were injected with an expression plasmid pSP SEAP coated with and without poly-L-glutamae.
- Figure 6 shows in vitro increased plasmid DNA stability when poly-L- glutamate is added to the solution. All samples were incubated for 6 month at 37°C.
- nucleic acid expression construct refers to any type of genetic construct comprising a nucleic acid coding for a RNA capable of being transcribed.
- expression vector can also be used interchangeably.
- the term "functional biological equivalent" of GHRH as used herein is a polypeptide that has been engineered to contain a distinct amino acid sequence while simultaneously having similar or improved biologically activity when compared to the GHRH polypeptide.
- encoded GHRH is a biologically active polypeptide.
- delivery as used herein is defined as a means of introducing a material into a subject, a cell or any recipient, by means of chemical or biological process, injection, mixing, electroporation, sonoporation, or combination thereof, either under or without pressure.
- subject refers to any species of the animal kingdom, hi preferred embodiments it refers more specifically to humans and animals used for: pets (e.g. cats, dogs, etc.); work (e.g. horses, cows, etc.); food (chicken, fish, lambs, pigs, etc); and all others known in the art.
- pets e.g. cats, dogs, etc.
- work e.g. horses, cows, etc.
- food chicken, fish, lambs, pigs, etc
- the term "recipient” as used herein refers to any species of the animal kingdom. In preferred embodiments it refers more specifically to humans and animals used for: pets (e.g. cats, dogs, etc.); work (e.g. horses, cows, etc.); food (chicken, fish, lambs, pigs, etc); and all others known in the art.
- promoter refers to a sequence of DNA that directs the transcription of a gene.
- a promoter may be "inducible", initiating transcription in response to an inducing agent or, in contrast, a promoter may be “constitutive”, whereby an inducing agent does not regulate the rate of transcription.
- a promoter may be regulated in a tissue-specific or tissue-preferred manner, such that it is only active in transcribing the operable linked coding region in a specific tissue type or types.
- coding region refers to any portion of the DNA sequence that is transcribed into messenger RNA ("mRNA”) and then translated into a sequence of amino acids characteristic of a specific polypeptide.
- analog includes any mutant of GHRH, or synthetic or naturally occurring peptide fragments of GHRH, as HV-GHRH (SEQID# 1), TI- GHRH (SEQID# 2), TV-GHRH (SEQID# 3), 15/27/28-GHRH (SEQID# 4), (1-44)NH 2 or (1- 40)OH (SEQID# 6) forms, or shorter forms to up to (1-29)NH 2 .
- growth hormone as used herein is defined as a hormone that relates to growth and acts as a chemical messenger to exert its action on a target cell.
- growth hormone releasing hormone As used herein is defined as a hormone that facilitates or stimulates release of growth hormone, and in a lesser extent other pituitary hormones, as prolactin.
- molecular switch refers molecule that is delivered into a subject that can regulate transcription of a gene.
- cassette as used herein is defined as one or more transgene expression vectors.
- post-injection refers to a time period following the introduction of a nucleic acid cassette that contains heterologous nucleic acid sequence encoding GHRH or biological equivalent thereof into the cells of the subject and allowing expression of the encoded gene to occur while the modified cells are within the living organism.
- plating refers to the positioning of a plurality of electrodes (either plate or needle) in a selected tissue.
- heterologous nucleic acid sequence as used herein is defined as a DNA sequence consisting of differing regulatory and expression elements.
- vector refers to any vehicle that delivers a nucleic acid into a cell or organism. Examples include plasmid vectors, viral vectors, liposomes, or cationic lipids. [0038] The term “electroporation” as used herein refers to a method that utilized electric pulses to deliver a nucleic acid sequence into cells.
- electrical pulse refers either a constant current pulse, or a constant-voltage pulse.
- PLG poly-L-glutamate
- spaced relationship refers to a positioning of electrodes in a tissue of a subject in either a symmetrical or non-symmetrical relationship to other electrodes.
- weight ratio refers to an amount of nucleic acid expression construct (in micrograms), to an amount of charged transfection-facilitating polypeptide (in micrograms) in a composition, regardless of the total volume delivered.
- mole ratio refers to an amount of nucleic acid expression construct (in moles), to an amount of charged transfection-facilitating polypeptide (in moles) in a composition.
- amino acids used herein are as follows: Alanine, A, ala; Arginine, R, arg; Asparagine, N, asn; Aspartic acid, N, asp; Cysteine, C, cys; Glutamine, Q, gin; Glutamic acid, E, glu; Glycine, G, gly; Histidine, H, his; Isoleucine, I, ile; Leucine, L, leu; Lysine, K, lys; Methionine, M, met; Phenylalanine, F, phe; Proline, P, pro; Serine, S, ser; Threonine, T, thr; Tryptophan, W, trp; Tyrosine, Y, tyr; Valine, V, val.
- composition formulations The ability of electroporation to enhance plasmid uptake into the skeletal muscle has been well documented, as described above.
- plasmid uptake for example, a plasmid formulated with transfection facilitating particles poly-L-glutamate (“PLG”) or polyvinylpyrolidone (“PVP”) has been observed to increase gene transfection and consequently increase gene expression to up to 10 fold into mice, rats and dog muscle.
- PLG poly-L-glutamate
- PVP polyvinylpyrolidone
- One aspect of the current invention is the combination of electroporation and transfection facilitating particles associated with nucleic acid expression constructs.
- PLG will increase the transfection of the plasmid during the electroporation process, not only by physically stabilizing the plasmid DNA, and facilitating the intracellular transport through the membrane pores, but also through an active transporting mechanism.
- Poly-L-glutamate is a stable compound, and resistant to high, denaturizing temperatures. PLG has been used previously to increase stability in vaccine preparations because it does not increase the vaccine's immunogenicity. Additionally, PLG has been used as an anti-toxin for post antigen inhalation or exposure top ozone. Plasmid DNA delivered by injection, electroporation, or both to the skeletal muscle are easily expressed, and can be measured as indicated by the physiologic levels of the transgene product in the circulation. Nevertheless, stabilization of naked DNA may be required and is necessary in some cases, as prolonged storage before usage, injection into a large number of animals.
- plasmid DNA may be stored in different temperature for variable periods of time, it is critical that plasmid solutions be stable for extended periods of time. It is important that the compound associated with the DNA is not toxic to the cells (e.g. muscle cells) and does not cause breakage of plasmid DNA. It would be preferable for the composition of plasmid DNA and associated transfection facilitating particles to have a similar or increased uptake into the target cells.
- This invention utilizes low concentrations (e.g. below 6 ⁇ g/ ⁇ l, preferably about 0.01 ⁇ g/ ⁇ l) of low and medium molecular weight poly-L-glutamate (e.g.
- nucleic acid expression vector 1-15 kDa, with an average of 10 kDa or 15-50 kDa, with an average of 35 kDa) compounds display all the desired properties for an effective composition of nucleic acid expression vector and transfection facilitating polypeptide.
- PLG can be used at a high concentration in non-electroporation applications, we have determined that low mole ratio of nucleic acid expression vector to PLG is optimum for electroporation applications to the skeletal muscle.
- An example of a useful mole ratio of nucleic acid expression vector to PLG is about 1 :5,000.
- Another example of a more useful mole ratio of nucleic acid expression vector to PLG comprises one about 1 :2,500.
- An example of a preferred mole ratio of nucleic acid expression vector to PLG is about 1 : 1 ,200.
- An illustrative mole ratio of nucleic acid expression vector to PLG comprises one about 1:800.
- a representative mole ratio of nucleic acid expression vector to PLG comprises one about 1 :500.
- An example of a select mole ratio of nucleic acid expression vector to PLG comprises one about 1 :200.
- Another example of an even more select mole ratio of nucleic acid expression vector to PLG comprises one about 1:100.
- An example of a preferential mole ratio of nucleic acid expression vector to PLG comprises one about 1:50.
- Another example of a more preferential mole ratio of nucleic acid expression vector to PLG comprises one about 1:20.
- An example of a even more preferential mole ratio of nucleic acid expression vector to PLG comprises one about 1:10.
- An example of a most preferred mole ratio of nucleic acid expression vector to PLG is about 1:1.
- the proper mole ratio can be calculated for the moles of an appropriately average length nucleic acid expression vector (e.g. in the range of 2,000 bp to 30,000 bp) to moles of PLG of low and medium molecular weight poly-L-glutamate (e.g. 1-15 kDa, with an average of 10 kDa or 15-50 kDa, with an average of 35 kDa).
- PLG of low and medium molecular weight poly-L-glutamate e.g. 1-15 kDa, with an average of 10 kDa or 15-50 kDa, with an average of 35 kDa.
- the pharmaceutical composition of the present invention may be delivered via various routes and to various sites in an animal body to achieve a particular effect.
- a particular route can provide a more immediate and more effective reaction than another route.
- local or systemic delivery can be accomplished by administration comprising application or instillation of the formulated composition into body cavities, inhalation or insufflation of an aerosol, or by parenteral introduction, comprising intramuscular, intravenous, peritoneal, subcutaneous, intradermal, as well as topical administration.
- different methods of delivery may be utilized to administer a plasmid/facilitating agent composition into a cell.
- Examples include: (1) methods utilizing physical means, such as electroporation (electricity), a gene gun (physical force) or applying large volumes of a liquid (pressure); and (2) methods wherein said vector is complexed to another entity, such as a liposome or transporter molecule.
- physical means such as electroporation (electricity), a gene gun (physical force) or applying large volumes of a liquid (pressure); and (2) methods wherein said vector is complexed to another entity, such as a liposome or transporter molecule.
- Constant Current Electroporation The underlying phenomenon of electroporation is believed to be the same in all cases, but the exact mechanism responsible for the observed effects has not been elucidated. Although not wanting to be bound by theory, the overt manifestation of the electroporative effect is that cell membranes become transiently permeable to large molecules, after the cells have been exposed to electric pulses. There are conduits through cell walls, which under normal circumstances, maintain a resting transmembrane potential of ca. 90 mV by allowing bi-directional ionic migration.
- electroporation makes use of the same structures, by forcing a high ionic flux through these structures and opening or enlarging the conduits.
- metallic electrodes are placed in contact with tissues and predetermined voltages, proportional to the distance between the electrodes are imposed on them.
- the electric field intensity E has been a very important value in prior art when formulating electroporation protocols for the delivery of a drug or macromolecule into the cell of the subject. Accordingly, it is possible to calculate any electric field intensity for a variety of protocols by applying a pulse of predetermined voltage that is proportional to the distance between electrodes.
- a caveat is that an electric field can be generated in a tissue with insulated electrodes (i.e. flow of ions is not necessary to create an electric field).
- it is the current that is necessary for successful electroporation not electric field per se.
- the heat produced is the product of the inter- electrode impedance, the square of the current, and the pulse duration. Heat is produced during electroporation in tissues and can be derived as the product of the inter-electrode current, voltage and pulse duration.
- the protocols currently described for electroporation are defined in terms of the resulting field intensities E, which are dependent on short voltage pulses of unknown current. Accordingly, the resistance or heat generated in a tissue cannot be determined, which leads to varied success with different pulsed voltage electroporation protocols with predetermined voltages.
- the ability to limit heating of cells across electrodes can increase the effectiveness of any given electroporation voltage pulsing protocol.
- Controlling the current flow between electrodes allows one to determine the relative heating of cells. Thus, it is the current that determines the subsequent effectiveness of any given pulsing protocol, and not the voltage across the electrodes. Predetermined voltages do not produce predetermined currents, and prior art does not provide a means to determine the exact dosage of current, which limits the usefulness of the technique. Thus, controlling an maintaining the current in the tissue between two electrodes under a threshold will allow one to vary the pulse conditions, reduce cell heating, create less cell death, and incorporate macromolecules into cells more efficiently when compared to predetermined voltage pulses.
- a constant-current electroporation device is the invention of a co-pending application entitled "Electrode assembly for constant current-electroporation and use” S/N 60/362,362 filed on March 7, 2002 with Westerstein et al, (“the Western '362 application”) listed as inventors, and is herby incorporated by reference.
- One aspect of the Western '362 application overcomes the above problem by providing a means to effectively control the dosage of electricity delivered to the cells in the inter-electrode space by precisely controlling the ionic flux that impinges on the conduits in the cell membranes.
- the precise dosage of electricity to tissues can be calculated as the product of the current level, the pulse length and the number of pulses delivered.
- the constant-current system comprises an electrode apparatus connected to a specially designed circuit, which is also utilized in the current invention.
- One aspect of the present invention is to provide a means to deliver the electroporative current to a volume of tissue along a plurality of paths without, causing excessive concentration of cumulative current in any one location, thereby avoiding cell death owing to overheating of the tissue.
- the composition of the nucleic acid expression vector associated with a transfection facilitation poly-peptide will further facilitate successful transfection protocols. For example, the maximal energy delivery from a particular pulse would occur along a line that connects two electrodes. Prior art teaches that the electrodes are present in pairs and that the voltage pulses are delivered to the paired electrodes of opposed polarity. Accordingly, the maximal energy delivery from a particular pulse would occur along a line that connects two electrodes.
- nucleic acid/transfection facilitation composition of the current invention can also help stabilize cells in prior art electroporation protocols.
- the electrodes of one embodiment of the present invention are arranged in a radial and symmetrical array, but unlike prior art, the electrodes are odd numbered, and not in opposing pairs ( Figure 2). Delivering an electric pulse to any two of the electrodes from an electric pulse generator results in a pattern that is best described as a polygon. Tracing this pattern would result in a five-point star with a pentagon of electrical pulses surrounding the center of the array in tissue where the concentration of molecules to be transfected is greatest. Although not wanting to be bound by theory, it is not the odd number of electrodes, per se, that makes a difference, but in the direction of the current paths. With the configuration of prior art, all the pulses generate a current that passes through the center of the assembly.
- the cumulated dose i.e. the heating effect is therefore concentrated in the center, with the peripheral dose falling off rapidly.
- the dose is spread more evenly, over a larger volume.
- the electrodes are arranged in an ⁇ array of five electrodes, the pulses might be sequentially applied to electrodes 1 and 3, then 3 and 5, then 5 and 2, then 2 and 4, then 4 and 1.
- the tissue between the electrodes is a volume conductor, a certain current intensity exists along parallel lines, weakening as the distance from the center line increases.
- the cumulative effect of a sequence of pulses results in a more uniform distribution of the energy delivered to the tissues, increasing the probability that the cells that have been electroporated actually survive the procedure.
- the nature of the voltage pulse to be generated is determine by the nature of tissue, the size of the selected tissue and distance between electrodes. It is desirable that the voltage pulse be as homogenous as possible and of the correct amplitude. Excessive field strength results in the lysing of cells, whereas a low field strength results in reduced efficacy of electroporation.
- Prior art inventions utilize the distance between electrodes to calculate the electric field strength and predetermined voltage pulses for electroporation. This reliance on knowing the distance between electrodes is a limitation to the design of electrodes. Because the programmable current pulse controller will determine the impedance in a volume of tissue between two electrodes, the distance between electrodes is not a critical factor for determining the appropriate electrical current pulse.
- an alternative embodiment of the needle electrode array design would be one that is non-symmetrical.
- one skilled in the art can imagine any number of suitable symmetrical and non-symmetrical needle electrode arrays that do not deviate from the spirit and scope of a particular electrode design.
- the depth of each individual electrode within an array and in the desired tissue could be varied with comparable results.
- multiple injection sites for the macromolecules could be added to the needle electrode array.
- the product of the measured inter- electrode impedance, the square of the current and the cumulated pulse duration is a measure of the total energy delivered.
- This quantity can be converted to degrees Celsius, when the volume of the tissues encompassed by the electrodes and the specific heat of the tissues are known.
- T the rise in tissue temperature
- R resistance
- I current
- Amperes current
- t length of pulse
- K conversion factor between joules and calories
- the advantage of a constant-current is that the pulse can be prevented from attaining an amplitude at which the cells are destroyed.
- the current can attain a destructive intensity, and the operator can not prevent that from happening, hi a constant-current system, the current is preset under a threshold level where cell death does not occur.
- the exact setting of the current is dependent of the electrode configuration, and it must be determined experimentally. However, once the proper level has been determined, cell survival is assured, from case to case.
- the addition of a nucleic acid expression construct associated with a transfection facilitating polypeptide increases the opportunity of electroporated cells to incorporate the plasmid construct.
- Nucleic acid constructs for therapy One aspect of this invention relates to a composition and method for efficient delivery of a nucleic acid construct to a tissue as a treatment for various diseases found in chronically ill subjects. More specifically, the aspects of this invention pertain to a method for delivering a heterologous nucleic acid sequence that is encoding a specific gene (e.g. growth hormone releasing hormone ("GHRH”) or biological equivalent thereof) into one or more cells of the subject (e.g. somatic, stem, or germ cells) and allowing expression of the encoded gene (e.g. GHRH or biological equivalent thereof) to occur while the modified cells are within the subject.
- GHRH growth hormone releasing hormone
- the method of delivering the nucleic acid sequence encoding the gene is via electroporation.
- the subsequent expression of the encoded gene can be regulated by a tissue specific promoter (e.g. muscle), and/or by a regulator protein that contains a modified ligand-binding domain (e.g. molecular switch), which will only be active when the correct modified ligand (e.g. mifepistone) is externally administered into the subject.
- a tissue specific promoter e.g. muscle
- a regulator protein that contains a modified ligand-binding domain
- the extracranial expression and ensuing release of GHRH or biological equivalent thereof by the modified cells can be used to treat anemia, wasting, immune dysfunction, life extension or other disorders in the chronically ill subject.
- Recombinant GH replacement therapy is widely used clinically, with beneficial effects, but generally, the doses are supraphysiological. Such elevated doses of recombinant GH are associated with deleterious side-effects, for example, up to 30% of the recombinant GH treated patients report a higher frequency of insulin resistance or accelerated bone epiphysis growth and closure in pediatric patients. In addition, molecular heterogeneity of circulating GH may have important implications in growth and homeostasis, which can lead to a less potent GH that has a reduced ability to stimulate the prolactin receptor.
- EXAMPLE 1 Plasmid vectors containing the muscle specific synthetic promoter SPc5-12 were previously described (Li et al., 1999). Wild type and mutated porcine GHRH cDNAs were generated by site directed mutagenesis of GHRH cDNA (Altered Sites II in vitro Mutagenesis System, Promega, Madison, WI), and cloned into the BamHI/ Hind III sites of pSPc5-12, to generate pSP-wt-GHRH, or pSP-HV-GHRH respectively. The 3' untranslated region (3'UTR) of growth hormone was cloned downstream of GHRH cDNA.
- the resultant plasmids contained mutated coding region for GHRH, and the resultant amino acid sequences were not naturally present in mammals.
- the effects on treating anemia; increasing total red blood cell mass in a subject; reversing the wasting; reversing abnormal weight loss; treating immune dysfunction; reversing the suppression of lymphopoesis; or extending life expectancy for the chronically ill subject are determined ultimately by the circulating levels of analog GHRH hormones.
- Several different plasmids that encoded different mutated amino acid sequences of GHRH or functional biological equivalent thereof are as follows:
- Ai is a D-or L-isomer of an amino acid selected from the group consisting of tyrosine ("Y"), or histidine ("H”
- a 2 is a D-or L-isomer of an amino acid selected from the group consisting of alanine ("A"), valine (“V"), or isoleucine ("I”)
- a 3 is a D-or L-isomer of an amino acid selected from the group consisting of alanine ("A”) or glycine (“G”)
- a 4 is a D-or L-isomer of an amino acid selected from the group consisting of methionein (“M”), or leucine (“L”)
- A5 is a D-or L-isomer of an amino acid selected from the group consisting of methionein ("M”), or leucine (“L”)
- A5 is a D-or L-isomer of an amino acid selected from the group consisting of methionein ("M”), or leucine
- Another plasmid that was utilized included the pSP-SEAP construct that contains the Sacl/ Hindlll SPc5-12 fragment, SEAP gene and SV40 3'UTR from pSEAP-2 Basic Vector (Clontech Laboratories, Inc., Palo Alto, CA).
- the plasmids described above do not contain polylinker, IGF-I gene, a skeletal ⁇ -actin promoter or a skeletal ⁇ -actin 3' UTR (untranslated region) / NCR (non- coding region). Furthermore, these plasmids were introduced by muscle injection, followed by in vivo electroporation, as described below. [0068] In terms of “functional biological equivalents,” it is well understood by the skilled artisan that, inherent in the definition of a "biologically functional equivalent" protein and/or polynucleotide, is the concept that there is a limit to the number of changes that may be made within a defined portion of the molecule while retaining a molecule with an acceptable level of equivalent biological activity.
- a peptide comprising a functional biological equivalent of GHRH is a polypeptide that has been engineered to contain distinct amino acid sequences while simultaneously having similar or improved biologically activity when compared to GHRH.
- one biological activity of GHRH is to facilitate growth hormone ("GH") secretion in the subject.
- mice Plasmid associated with PLG in mice.
- pSP-SEAP approximately 5,000 base pairs
- the mole ratios are provided for the purpose of example.
- the mole ratio listed in Table 1 are based upon a 5,000 bp nucleic acid expression vector, and PLG with the weighted average molecular weight of 10,900.
- group 2 in Table 1 has an injection total of 30 ⁇ g of DNA vector associated with 150 ⁇ g of transfection facilitating polypeptides, wherein the mole ratio is 1:1,200.
- group 3 in Table 1 has an injection total of 30 ⁇ g of DNA vector associated with 0.25 ⁇ g of transfection facilitating polypeptides, wherein the mole ratio is less than 1:2.
- Mole ratio's of DNA vector to PLG having a 1:1 relationship comprises a lower limit formulation still having a higher transfection efficiency than a "naked" DNA vector alone.
- One of ordinary skill in the art is capable of formulating mole ratio calculations with different length expression vectors and variable molecular weights of PLG. Additionally, it is understood that the length of the nucleic acid expression vector and the weighted average molecular weight of PLG is subject to change based upon specific vector lengths and particular formulation strategies known to one skilled in the art (e.g. functional nucleic acid expression vectors greater than or less than about 5,000 nucleotides, and PLG having an average molecular weight of less than about 1 to about 30 kDa). Consequently, even the smallest of PLG polymers (e.g. PLG trimers having a molecular weight ⁇ 400 Da) can be used for this invention.
- PLG polymers e.g. PLG trimers having a molecular weight ⁇ 400 Da
- Electroporation was carried out using a constant current electroporation apparatus that is the subject of the Western '362 co-pending application. This device was used to deliver square wave pulses in all experiments. The amplitude conditions of 1 mA, 5 pulses, 50 milliseconds per pulse were used. Caliper electrodes were used to deliver in vivo electric pulses. The caliper (plate) electrodes consisted of 1.5 cm square metallic blocks mounted on a ruler, so the distance between the plates could be easily assessed. Plasmid DNA or associated DNA was injected through the intact skin into the tibialis anterior muscle of mice. Each animal received one injection into a single injection site.
- mice were bled and serum collected for up to 3 month post-injection.
- the SEAP molecule usually disappears after birth, and it is immunogenic in adult animals.
- Blood was collected by tail vein collection for mice, before plasmid administration, and up to 3 month post-injection in mice.
- Serum levels of SEAP were determined using a chemiluminescence assay (Tropix, Bedford, MA) following the manufacturer instructions.
- Figure 3 shows the serum SEAP levels for all five groups of mice described in Table 1.
- mice from group 5 i.e. nucleic acid expression construct coated with 0.01 ⁇ g/ ⁇ l PLG
- group 2 injected with plasmid associated with 6 ⁇ g/ ⁇ l had high inflammation and some morphological changes. This observation correlates with the data in the literature, that shows short-term enhanced expression using PLG compounds, expression that disappears at approximately 1 month post-injection. (Fewell et al., 2001).
- EXAMPLE 2 - PLG coating in pigs In order to demonstrate similar results in a larger mammal, experiments similar to Example 1 above were conducted in pigs. Thus, two groups of three pigs were injected with 500 ⁇ g (micrograms) of pSP-SEAP and electroporated. The plasmid expressed secreted embryonic alkaline phosphatase ("SEAP"). The molecule usually disappears after birth, and it is immunogenic in adult animals. One group received naked nucleic acid construct and the second group received the nucleic acid construct in 0.01 ⁇ g/ ⁇ l PLG Pigs were weighted and bled prior to injection, and every other day up to 10 days post-injection.
- SEAP embryonic alkaline phosphatase
- Electroporation devices A constant current electroporator machine (Advisys, Inc.) was used to deliver square wave pulses in all experiments. The electroporation parameters included an amplitude condition of 1 mA, 5 pulses, 50 milliseconds per pulse. A needle electrode was used to deliver in vivo electric pulses.
- the 5-needle electrode device consists of a circular array (1 cm diameter) of equally spaced filled 21 -gauge needles mounted on a non-conductive material. All needles were 2 cm in length and during all injections or electroporations, the needles were completely inserted into the muscle. Plasmid DNA was injected through the intact skin into the semitendinosous muscle of pigs with a 21 g needle. Each animal received one injection into a single injection site and the injection site also received a tattoo so it could be easily isolated at the end of the experiment.
- Example 3 PLG coating in Dogs : In order to demonstrate similar results in a different species of larger mammal, experiments similar to Example 2 were conducted in dogs. Thus, a comparison of expression in dogs injected with 5 needle array electrodes, with coated or naked plasmid.
- Four groups of 5 dogs were injected with a plasmid DNA, pSP- SEAP, expressing the secreted embryonic alkaline phosphatase ("SEAP").
- SEAP embryonic alkaline phosphatase
- the molecule usually disappears after birth, and it is immunogenic in adult animals. No adverse reaction, or change in biochemical, clinical or hormonal profiles is related to the development of the immune response to SEAP in animals.
- the injection was followed by electroporation, using standard conditions, and 5 needle electrodes.
- the plasmid DNA was either naked, or coated with a mol / mol dilution of poly-L-glutamate.
- the groups are as follows:
- Dogs were weight and bled at baseline (pre-inj ection) and every other day to day 10 post-injection. Serum was assayed for SEAP. Values were corrected for weight (blood volume). SEAP values were analyzed for differences in between the different injected groups. The results of these experiments are shown in Figure 5. The results showed that a 5 needle electrode could be used in dogs to efficiently mediate electroporation. Additionally, PLG coated DNA increasing plasmid stability and electroporation efficiency in dogs.
- Example 4 PLG increases plasmid stability in vitro at high temperatures: In order to evaluate the effects of the PLG on plasmid stability, the following assay has been performed. Plasmid pSP-HV-GHRH encoding for a super-porcine growth hormone releasing hormone was diluted into distilled water to a final concentration of 2 mg/ml. PLG in a mol / mol ratio was added to a group of samples, while PLG was not added into control samples. All samples were incubated for 6 month at 37°C. After 6 month, aliquots were taken from all samples, and run onto an agarose gel (Figure 6). As seen in the gel image, all plasmid is present in the samples were PLG was added, while in control samples all plasmid is completely degraded.
- the present invention provides a method of transferring a therapeutic gene to a host, which comprises administering the vector of the present invention, preferably as part of a composition, using any of the aforementioned routes of administration or alternative routes known to those skilled in the art and appropriate for a particular application.
- Effective gene transfer of a vector to a host cell in accordance with the present invention to a host cell can be monitored in terms of a therapeutic effect (e.g.
- alleviation of some symptom associated with the particular disease being treated or, further, by evidence of the transferred gene or expression of the gene within the host (e.g., using the polymerase chain reaction in conjunction with sequencing, Northern or Southern hybridizations, or transcription assays to detect the nucleic acid in host cells, or using immunoblot analysis, antibody-mediated detection, mRNA or protein half-life studies, or particularized assays to detect protein or polypeptide encoded by the transferred nucleic acid, or impacted in level or function due to such transfer).
- evidence of the transferred gene or expression of the gene within the host e.g., using the polymerase chain reaction in conjunction with sequencing, Northern or Southern hybridizations, or transcription assays to detect the nucleic acid in host cells, or using immunoblot analysis, antibody-mediated detection, mRNA or protein half-life studies, or particularized assays to detect protein or polypeptide encoded by the transferred nucleic acid, or impacted in level or function due to such transfer).
- compositions can be further approximated through analogy to compounds known to exert the desired effect.
- the actual dose and schedule can vary depending on whether the compositions are administered in combination with other pharmaceutical compositions, or depending on inter-individual differences in pharmacokinetics, drug disposition, and metabolism.
- amounts can vary in in vitro applications depending on the particular cell line utilized (e.g., based on the number of vector receptors present on the cell surface, or the ability of the particular vector employed for gene transfer to replicate in that cell line).
- the amount of vector to be added per cell will likely vary with the length and stability of the therapeutic gene inserted in the vector, as well as the nature of the sequence, and is particularly a parameter which needs to be determined empirically, and can be altered due to factors not inherent to the methods of the present invention (for instance, the cost associated with synthesis).
- One skilled in the art can easily make any necessary adjustments in accordance with the exigencies of the particular situation.
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Abstract
Description
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US15667002A | 2002-05-28 | 2002-05-28 | |
US156670 | 2002-05-28 | ||
US395709 | 2003-03-24 | ||
US10/395,709 US20040014645A1 (en) | 2002-05-28 | 2003-03-24 | Increased delivery of a nucleic acid construct in vivo by the poly-L-glutamate ("PLG") system |
PCT/US2003/016541 WO2003099341A1 (en) | 2002-05-28 | 2003-05-23 | Increased delivery of a nucleic acid constrtuct in vivo by the poly-l-glutamate (plg) system |
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CN (1) | CN1662261A (en) |
AU (1) | AU2003273142A1 (en) |
BR (1) | BR0311539A (en) |
CA (1) | CA2485976A1 (en) |
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JPWO2005089810A1 (en) * | 2004-03-22 | 2008-01-31 | 関西ティー・エル・オー株式会社 | Bone induction method by introducing human bone morphogenetic gene using electroporation method |
WO2006014737A2 (en) * | 2004-07-23 | 2006-02-09 | Advisys, Inc. | Growth hormone releasing hormone enhances the immune response induced by vaccination |
Citations (3)
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WO2001006988A2 (en) * | 1999-07-26 | 2001-02-01 | Baylor College Of Medicine | Super-active porcine growth hormone releasing hormone analog |
WO2001066149A2 (en) * | 2000-03-03 | 2001-09-13 | Valentis, Inc. | Nucleic acid formulations for gene delivery and methods of use |
WO2003049700A2 (en) * | 2001-12-11 | 2003-06-19 | Advisys, Inc. | Growth hormone releasing hormone suplementation for treating chronically ill subjects |
-
2003
- 2003-05-23 BR BR0311539-9A patent/BR0311539A/en not_active Application Discontinuation
- 2003-05-23 AU AU2003273142A patent/AU2003273142A1/en not_active Abandoned
- 2003-05-23 EP EP03741818A patent/EP1513559A4/en not_active Withdrawn
- 2003-05-23 MX MXPA04011766A patent/MXPA04011766A/en not_active Application Discontinuation
- 2003-05-23 WO PCT/US2003/016541 patent/WO2003099341A1/en not_active Application Discontinuation
- 2003-05-23 CA CA002485976A patent/CA2485976A1/en not_active Abandoned
- 2003-05-23 CN CN 03814625 patent/CN1662261A/en active Pending
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WO2001006988A2 (en) * | 1999-07-26 | 2001-02-01 | Baylor College Of Medicine | Super-active porcine growth hormone releasing hormone analog |
WO2001066149A2 (en) * | 2000-03-03 | 2001-09-13 | Valentis, Inc. | Nucleic acid formulations for gene delivery and methods of use |
WO2003049700A2 (en) * | 2001-12-11 | 2003-06-19 | Advisys, Inc. | Growth hormone releasing hormone suplementation for treating chronically ill subjects |
Non-Patent Citations (3)
Title |
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DRAGHIA-AKLI R ET AL: "ELECTRICAL ENHANCEMENT OF FORMULATED PLASMID DELIVERY IN ANIMALS" TECHNOLOGY IN CANCER RESEARCH AND TREATMENT, ADENINE PRESS, SCHENECTADY, NY, US, vol. 1, no. 5, October 2002 (2002-10), pages 365-371, XP001182428 ISSN: 1533-0346 * |
FEWELL J G ET AL: "Gene therapy for the treatment of hemophilia B using PINC-formulated plasmid delivered to muscle with electroporation" MOLECULAR THERAPY, ACADEMIC PRESS, SAN DIEGO, CA,, US, vol. 3, no. 4, April 2001 (2001-04), pages 574-583, XP002965532 ISSN: 1525-0016 * |
See also references of WO03099341A1 * |
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WO2003099341A1 (en) | 2003-12-04 |
CA2485976A1 (en) | 2003-12-04 |
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AU2003273142A1 (en) | 2003-12-12 |
CN1662261A (en) | 2005-08-31 |
EP1513559A4 (en) | 2006-01-18 |
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