 |
Description  |
|
|
INTRODUCTION
1. Technical Field
The present invention relates to methods and compositions for producing a transgenic mammal which comprises an exogenously supplied gene in lung tissue. The gene is supplied by aerosolized delivery, particularly to the airways and alveoli of the
lung.
2. Background
With the advent of molecular cloning techniques, an expanding array of genes with mutations responsible for important human diseases have been identified and isolated. To date, attempts to replace absent or mutated genes in human patients have
relied on ex vivo techniques. Ex vivo techniques include, but are not limited to, transformation of cells in vitro with either naked DNA or DNA encapsulated in liposomes, followed by introduction into a host organ ("ex vivo" gene therapy). The criteria
for a suitable organ include that the target organ for implantation is the site of the relevant disease, the disease is easily accessible, that it can be manipulated in vitro, that it is susceptible to genetic modification methods and ideally, it should
contain either non-replicating cells or cycling stem cells to perpetuate a genetic correction. It also should be possible to reimplant the genetically modified cells into the organism in a functional and stable form. A further requirement for ex vivo
gene therapy, if for example a retroviral vector is used, is that the cells be pre-mitotic; post-mitotic cells are refractory to infection with retroviral vectors. Exemplary of a target organ which meets the criteria for in vitro gene transfer is the
mammalian bone marrow.
There are several drawbacks to ex vivo therapy. For example, if only differentiated, replicating cells are infected, the newly introduced gene function will be lost as those cells mature and die. Ex vivo approaches also can be used to transfect
only a limited number of cells and cannot be used to transfect cells which are not first removed from the body.
Retroviruses, adenoviruses and liposomes have been used in animal model studies in attempts to increase the efficiency of gene transfer; DNA has been introduced into animals by intratracheal (IT), intravenous, intraperitoneal, intramuscular, and
intraarterial injection. Expression of introduced genes, either complexed to cationic vectors or packaged in adenoviral vectors has been demonstrated in the lungs of rodents after IT instillation. However, IT injection is invasive and produces a
non-uniform distribution of the instilled material; it also is too invasive to be performed repeatedly in humans. It therefore would be of interest to develop a non-invasive delivery technique which also results in deeper penetration of material into
the lung than other methods, and can be used to deposit material evenly throughout the airways and alveoli. Such a delivery technique could be used as a means of treatment for genetic disorders, particularly of the lung, via generalized transgene
expression in lung cells in vivo.
Relevant Literature
Hazinski, et al., Am. J. Respir. Cell Mol. Biol. (1991) 4: 206-209, relates to liposome-mediated gene transfer of DNA into the intact rodent lung. Three fusion gene constructs were complexed to cationic liposomes including (1) the
chloramphenicol acetyltransferase ("CAT") gene linked to a Rous sarcoma virus ("RSV") promoter; (2) the CAT gene linked to a mouse mammary tumor virus ("MMTV") promoter; and (3) a cytomegalovirus-.beta.-galactosidase ("CMV-.beta.-gal") fusion gene. The
liposome/DNA complexes were instilled into the cervical trachea of rats and detectable levels of gene expression observed.
Brigham et al., Am. J. Med. Sci. (1989) 298: 278-281, describes the in vivo transfection of murine lungs with the CAT gene using a liposome vehicle. Transfection was accomplished by intravenous, intratracheal or intraperitoneal injection.
Both intravenous and intratracheal administration resulted in the expression of the CAT gene in the lungs. However, intraperitoneal administration did not. See, also Werthers, Clinical Research (1991) 39: (Abstract).
Canonico et al., Clin. Res. (1991) 39: 219A describes the expression of the human .alpha.-1 antitrypsin gene, driven by the CMV promoter, in cultured bovine lung epithelial cells. The gene was added to cells in culture using cationic liposomes. The experimenters also detected the presence of .alpha.-1 antitrypsin in histological sections of the lung of New Zealand white rabbits following the intravenous delivery of gene constructs complexed to liposomes. Yoshimura et al. disclose expression of
the human cystic fibrosis transmembrane conductance regulator gene in mouse lung after intratracheal liposome-DNA gene transfer. Wolff et al., Science (1990) 247: 1465-1468 relates to direct transfer of the CAT, .beta.-gal and luciferase genes into
mouse skeletal muscle in vivo. Gene expression was observed in all three cases. Nabel et al., Science (1990) 249: 1285-1288, pertains to in vivo intra-arterial transfection of pigs with liposomes containing a .beta.-gal expression plasmid.
Site-specific gene expression was observed in the arterial wall. None of the above cited art, however, practices or teaches the use of aerosol administration to deliver genes directly to the lung. An example of a review article of human gene therapy
procedures is: Anderson, Science (1992) 256: 808-813.
PCT/US90/01515, having International Publication No. WO 90/11092, describes a method for introducing naked DNA into muscle tissue. Debs et al. disclose pentamidine uptake in the lung by aerosolization and delivery in liposomes. Am Rev Respir
Dis (1987) 135: 731-737.
SUMMARY
Methods and compositions are provided for producing a mammal which comprises an exogenously supplied nucleic acid in its lung cells. The method includes the steps of preparing a lipid carrier-nucleic acid mixture suitable for nebulization,
nebulizing the mixture, and depositing the resulting nebulized mixture in the lung of a mammalian host of interest in an amount sufficient to transform cells contacted by the deposited nebulized mixture. The exogenously supplied nucleic acid generally
is provided in an expression cassette and includes a coding sequence operably joined to transcriptional and translational regulatory sequences functional in the mammal. The methods and compositions find use particularly for in vivo gene therapy of
pulmonary disorders.
BRIEF DESCRIPTION OF THE FIGURES
The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawing(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee.
FIG. 1 demonstrates that aerosol administration of pRSV-CAT-DOTMA: cholesterol complexes resulted in expression of the CAT gene in mouse lungs. Lanes 1-3 were derived from mice receiving no treatment; lanes 4-6 represent mice administered 0.5 mg
pRSV-CAT with 1.0 .mu.mole DOTMA-cholesterol liposomes; lanes 7-9 were derived from mice receiving 2.0 mg pRSV-CAT alone; and lanes 10-12 represent mice given 2.0 mg pRSV-CAT with 4.0 .mu.mol DOTMA-cholesterol liposomes in a 2 to 1 molar ratio. The CAT
gene is not normally present in mammalian cells; lanes 10-12 show spots indicative of CAT activity (the positive spots in lanes 10-11 are faint and do not reproduce well in the figure). The results thus indicate that the lung was successfully
transfected by the pRSV-CAT DOTMA-cholesterol:liposome aerosol. The results also show that neither aerosol administration of the pRSV-CAT alone, nor a lower aerosol dose of pRSV-CAT: DOTMA-cholesterol complexes produce detectable expression of the CAT
gene in mouse lungs. Thus, both the cationic liposome carrier, and a sufficient dose of DOTMA: liposome-RSV-CAT DNA complexes are required to produce transgene expression in the lung after aerosol administration. Maximum transgene expression is
achieved by complexing the liposomes and DNA together at an appropriate ratio and in an appropriate diluent.
FIG. 2 shows the results of an experiment where mice were administered 12 mg of pCIS-CAT complexed to 24 .mu.moles of DOTMA/DOPE 1:1 liposomes. Lanes 1-3 show the results from animals administered the aerosol in an Intox-designed nose-only
aerosol exposure chamber; lanes 4-7 are derived from mice exposed to the aerosol in a modified mouse cage; and lanes 8-10 show the results from animals placed in a smaller modified cage after being put in restrainers originally constructed for use in the
Intox chamber.
FIGS. 3A, 3B, and 3C show construction of pZN13.
FIGS. 4A and 4B show construction of pZN29.
FIG. 5 show construction of pZN32.
FIGS. 6A-6F show the results of immunostaining for intracellular CAT protein in lung sections from mice sacrificed 72 hours after receiving an aerosol containing 12 mg of pCIS-CAT plasmid complexed to 24 .mu.mols of DOTMA:DOPE liposomes (6A, 6B,
6C, 6D), or from untreated mice (6E, 6F). The section shown in 6D was treated with normal rabbit serum in place of anti-CAT antibody. Magnification: 6A, 6D (.times.50); 6B, 6C, 6E (.times.250).
FIG. 7 shows CAT activity in lung extracts from mice sacrificed 72 hours after receiving an aerosol containing either 12 mg of CMV-CAT plasmid alone or 12 mg of CMV-CAT plasmid complex to 24 .mu.mols of DOTMA:DOPE (1:1) liposomes. Untreated mice
were also assayed.
FIGS. 8A and 8B: FIG. 8A shows CAT activity in lung extracts from mice sacrificed from one to twenty-one days after receiving an aerosol containing 12 mg of pCIS-CAT plasmid complexed to 24 .mu.mols of DOTMA:DOPE liposomes; and FIG. 8B shows CAT
activity in several different tissue extracts from mice and indicates that expression of the transgene is lung-specific after aerosolization of DNA-liposome complexes into normal mice sacrificed at the three day time point in FIG. 8A. Control extract
contains purified CAT enzyme.
FIG. 9 shows Southern blot hybridization of genomic DNA from the lungs of mice sacrificed immediately after receiving an aerosol containing 12 mg of pCIS-CAT plasmid complexed to 24 .mu.mols of DOTMA:DOPE liposomes (lanes 1-4, 6-9) and from an
untreated control mouse (lane 5). Samples were digested with the restriction enzyme HindIII and probed with a 1.6 kb CAT fragment (upper panel). The same membrane was hybridized with a 1.1 kb BSU 36-1 single copy probe from a mouse factor VIII. A
genomic clone (lower panel).
FIGS. 10A-10E show photomicrographs of frozen sections from lungs of control mice (FIGS. 10B and 10D) and mice treated with pZN32 complexed to DDAB:cholesterol (1:1) liposomes (FIGS. 10A, 10C, and 10E).
FIGS. 11A(1), 11A(2), 11A(3), 11A(4), 11A(5), 11A(6), 11A(7), 11A(8), and 11A(9) show a full restriction map of the immediate early enhancer and promoter region for HCMV (Towne).
FIGS. 11C(1), 11C(2), 11C(3), 11C(4), 11C(5), 11C(6), and 11C(7) show a full restriction map of the immediate early enhancer and promoter region for HCMV (AD169).
FIGS. 11B(1), 11B(2), 11B(3), 11B(4), 11B(5), 11B(6) show a sequence comparison of the two HCMV promoters. The position of the NcoI site is indicated by an asterisk.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
In accordance with the subject invention, nucleic acid constructs together with methods of preparation and use are provided which allow for in vivo modulation of phenotype and/or genotype of cells in the respiratory tract of a mammalian host
following delivery of a sufficient dose of a lipid carrier-nucleic acid aerosol to the host mammal to provide for transfection of host lung cells. The lipid carrier-nucleic acid aerosol is obtained by nebulization of a lipid carrier-nucleic acid sample
mixture prepared in a biologically compatible fluid that minimizes aggregation of the lipid carrier-nucleic acid complexes. The methods and compositions can be used to produce a mammal comprising an exogenously supplied gene in lung tissue, particularly
alveolar and airway passage cells.
Central to the present invention is the discovery that genes can be delivered to the lung via aerosol administration, and subsequently expressed in vivo. The instant invention takes advantage of the use of lipid carriers as a delivery mechanism. Lipid carriers are able to stably bind through charge interactions or entrap and retain nucleic acid and permit a system amenable to nebulization, whereby intact genes can be delivered to specific pulmonary tissues. Lipid carriers include but are not
limited to liposome and micellas, as well as biodegradable cationic compounds comprising modified phosphoglycerides particularly alkylphosphoglycerides. Particular sites in the lung are targeted by varying the size of the aerosol particles administered,
as discussed more fully below. Targeting agents, such as antibodies directed against surface antigens expressed on specific pulmonary cell types, can also be covalently conjugated to the lipid carrier surface so that nucleic acid can be delivered to
specific cell types. Lipid carriers also allow for the delivery of relatively large mounts of nucleic acid, without a toxic effect, such that therapeutically effective mounts of the desired protein can be expressed in vivo. For a review of the use of
liposomes as carriers for delivery of nucleic acids, see, Hug and Sleight, Biochim. Biophys. Acta. (1991) 1097: 1-17; Straubinger et ed., in Methods of Enzymology (1983), Vol. 101, pp. 512-527.
Lipid carriers, particularly liposomes, have been used effectively, particularly to introduce drugs, radiotherapeutic agents, enzymes, viruses, transcription factors and other cellular effectors into a variety of cultured cell lines and animals.
In addition, successful clinical trials examining the effectiveness of liposome-mediated delivery of small drug molecules and peptides which act extracellularly have been reported. However, while the basic methodology for using liposome-mediated vectors
is well developed and has been shown to be safe, the technique previously has not been developed for aerosolized delivery of nucleic acid to pulmonary tissue for in vivo gene therapy. By in vivo gene therapy is meant transcription and/or translation of
exogenously supplied nucleic acid sequences to prevent, palliate and/or cure animal or human disease.
In addition to the discovery that transformation of lung cells can be obtained using aerosolized lipid carrier-nucleic acid complexes, several factors have been identified that can affect the relative ability of particular lipid carrier-nucleic
acid complexes to provide transformation of lung cells following aerosolized delivery of a solution containing the lipid carrier-nucleic acid constructs and to achieve a high level of expression where that is the desired endpoint. The factors include
(1) preparation of a solution that prior to or during nebulization will not form macroaggregates and wherein the nucleic acid is not sheared into fragments and (2) preparation both of lipid carriers and of expression constructs that provide for
predictable transformation of host lung cells following aerosolization of the lipid carrier-nucleic acid complex and administration to the host animal. Other factors include the lipid carrier:nucleic acid ratio in the solution for nebulization and the
diluent used to prepare the solution. These factors are discussed in detail below.
Aerosol delivery of nucleic acid-lipid carrier complexes provides a number of advantages over other modes of administration. For example, aerosol administration can serve to reduce host toxicity. Such an effect has been observed with the
delivery of substances such as pentamidine and cytokines, which can be highly toxic when delivered systematically, but are well tolerated when aerosolized. Additionally, the results in rodents with aerosolized pentamidine accurately predicted results in
human patients with AIDS treated with aerosolized pentamidine. See, for example, Debs et al., Antimicrob. Agents Chemother. (1987) 31: 37-41; Debs et al., Amer. Rev. Respir. Dis. (1987)135: 731-737; Debs et al., J. Immunol. (1988) 140: 3482-3488;
Montgomery et al., Lancet (1987) 11: 480-483; Montgomery et al., Chest (1989) 95: 747-751; Leoung et al., N. Eng. J. Med. (1990) 323: 769-775. Additionally, rapid clearance of circulating liposomes by the liver and spleen reticuloendothelial system is
avoided, thereby allowing the sustained presence of the administered substance at the site of interest, the lung. Serum induced inactivation of the therapeutic agent is also reduced. This method of transfection lung cells also avoids exposure of the
host mammal's gonads, thus avoiding transfection of germ line cells.
Other advantages of the subject invention include ease of administration i.e., the host mammal simply inhales the aerosolized lipid carrier-nucleic acid solution into the intended tissue, the lung. Further, by varying the size of the nebulized
particles some control may also be exercised over where in the lung the aerosol is delivered. Delivery may be extended over a long time period. Thus, there is a significant increase in the time period that target cells are exposed to the expression
constructs. Distribution of the aerosol is even throughout areas of the lung accessible to the spray. These advantages are significant, particularly when compared to other routes of administration such as intratracheal delivery which is invasive, the
expression constructs are delivered in a bolus which may disrupt the mucous barrier and additionally may result in pooling of the introduced fluid in areas of the lung at lower elevation. Further, damage from insertion of the intratracheal tube may
alter the ability of cells coming into contact with the expression constructs to be transfected.
The type of vector used in the subject application may also be an advantage. For example, most gene therapy strategies have relied on transgene insertion into retroviral or DNA virus vectors. Potential disadvantages of retrovirus vectors, as
compared to the use of lipid carriers, include the limited ability of retroviruses to mediate in vivo (as opposed to ex vivo) transgene expression; the inability of retrovirus vectors to transfect non-dividing cells; possible recombination events in
replication-defective retrovirus vectors, resulting in infectious retroviruses; possible activation of oncogenes or inhibition of tumor suppressor genes due to the random insertion of the transgene into host cell genomic DNA; size limitations (less than
15 kb of DNA can be packaged in a retrovirus vector, whereas lipid carriers can be used to deliver sequences of DNA of.gtoreq.250 kb to mammalian cells) and potential immunogenicity of the viral vectors leading to a host immune response against the
vector. In addition, all ex vivo approaches require that the cells removed from the body be maintained in culture for a period of time. While in culture, cells may undergo deleterious or potentially dangerous phenotypic and/or genotypic changes.
Adenovirus and other DNA viral vectors share several of the above potential limitations. Particularly for human use, but also for repeated veterinary use, biodegradable lipid carriers may be used which are metabolized by the host mammal to naturally
occurring compounds that are non-toxic to the host and/or are readily excreted.
The nucleic acid constructs generally will be provided as expression cassettes which will include as operably linked components in the direction of transcription, a transcriptional initiation region, a nucleic acid sequence of interest and a
transcriptional termination region wherein the transcriptional regulatory regions are functional in the mammalian host lung cell. An intron optionally may be included in the construct, preferably.gtoreq.100 bp and placed 5' to the coding sequence.
Generally it is preferred that the construct not become integrated into the host cell genome and it is introduced into the host as part of a non-integrating expression cassette. A coding sequence is "operably linked to" or "under the control of"
transcriptional and/or translational regulatory regions in a cell when DNA polymerase will bind the promoter sequence and transcribe the coding sequence into mRNA, either a sense strand or an antisense strand. Thus, the nucleic acid sequence includes
DNA sequences which encode polypeptides which are directly or indirectly responsible for a therapeutic effect, as well as genes coding for active nucleotide sequences such as antisense sequences and ribozymes.
The constructs for use in the invention include several forms, depending upon the intended use of the construct. Thus, the constructs include vectors, trancriptional cassettes, expression cassettes and plasmids. The transcriptional and
translational initiation region (also sometimes referred to as a "promoter,"), preferably comprises a transcriptional initiation regulatory region and a translational initiation regulatory region of untranslated 5' sequences, "ribosome binding sites,"
responsible for binding mRNA to ribosomes and translational initiation. It is preferred that all of the transcriptional and translational functional elements of the initiation control region are derived from or obtainable from the same gene. In some
embodiments, the promoter will be modified by the addition of sequences, such as enhancers, or deletions of nonessential and/or undesired sequences. By "obtainable" is intended a promoter having a DNA sequence sufficiently similar to that of a native
promoter to provide for the desired specificity of transcription of a DNA sequence of interest. It includes natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences.
For the transcriptional initiation region, or promoter element, any region may be used with the proviso that it provides the desired level of transcription of the DNA sequence of interest. The transcriptional initiation region may be native to
or homologous to the host cell, and/or to the DNA sequence to be transcribed, or foreign or heterologous to the host cell and/or the DNA sequence to be transcribed. By foreign to the host cell is intended that the transcriptional initiation region is
not found in the host into which the construct comprising the transcriptional initiation region is to be inserted. By foreign to the DNA sequence is intended a transcriptional initiation region that is not normally associated with the DNA sequence of
interest. Efficient promoter elements for transcription initiation include the SV40 (simian virus 40) early promoter, the RSV (Rous sarcoma virus) promoter, the Adenovirus major late promoter, and the human CMV (cytomegalovirus) immediate early 1
promoter.
Inducible promoters also find use with the subject invention where it is desired to control the timing of transcription. Examples of promoters include those obtained from .beta.-inteferon gene, a heat shock gene, a metallothionein gene or those
obtained from steroid hormone-responsive genes, including insect genes such as that encoding the ecdysone receptor. Such inducible promoters can be used to regulate transcription of the transgene by the use of external stimuli such as inteferon or
glucocorticoids. Since the arrangement of eukaryotic promoter elements is highly flexible, combinations of constitutive and inducible elements also can be used. Tandem arrays of two or more inducible promoter elements may increase the level of
induction above baseline levels of transcription which can be achieved when compared to the level of induction above baseline which can be achieved with a single inducible element.
Generally, the regulatory sequence comprises DNA up to about 1.5 Kb 5' of the transcriptional start of a gene, but can be significantly smaller. This regulatory sequence may be modified at the position corresponding to the first codon of the
desired protein by site-directed mutagenesis Kunkel TA, 1985, Proc. Natl. Acad. Sci. (USA), 82: 488-492) or by introduction of a convenient linker oligonucleotide by ligation, if a suitable restriction site is found near the N-terminal codon. In the
ideal embodiment, a coding sequence with a compatible restriction site may be ligated at the position corresponding to codon #1 of the gene. This substitution may be inserted in such a way that it completely replaces the native coding sequence and thus
the substituted sequence is flanked at its 3' end by the gene terminator and polyadenylation signal.
Transcriptional enhancer elements optionally may be included in the expression cassette. By transcriptional enhancer elements is intended DNA sequences which are primary regulators of transcriptional activity and which can act to increase
transcription from a promoter element, and generally do not have to be in the 5' orientation with respect to the promoter in order to enhance transcriptional activity. The combination of promoter and enhancer element(s) used in a particular expression
cassette can be selected by one skilled in the art to maximize specific effects. Different enhancer elements can be used to produce a desired level of transgene expression in a wide variety of tissue and cell types. For example, the human CMV immediate
early promoter-enhancer element can be used to produce high level transgene expression in many different tissues in vivo.
Examples of other enhancer elements which confer a high level of transcription on linked genes in a number of different cell types from many species include enhancers from SV40 and RSV-LTR. The SV40 and RSV-LTR are essentially constitutive.
They may be combined with other enhancers which have specific effects, or the specific enhancers may be used alone. Thus, where specific control of transcription is desired, efficient enhancer elements that are active only in a tissue-, developmental-,
or cell-specific fashion include immunoglobulin, interleukin-2 (IL-2) and .beta.-globin enhancers are of interest. Tissue-, developmental-, or cell-specific enhancers can be used to obtain transgene expression in particular cell types, such as
B-lymphocytes and T-lymphocytes, as well as myeloid, or erythroid progenitor cells. Alternatively, a tissue-specific promoter such as that derived from the human cystic fibrosis transmembrane conductance regulator (CFTR) gene can be fused to a very
active, heterologous enhancer element, such as the SV40 enhancer, in order to confer both a high level of transcription and tissue-specific transgene transcription. In addition, the use of tissue-specific promoters, such as LCK, may allow targeting of
transgene transcription to T lymphocytes. Tissue specific transcription of the transgene may be important, particularly in cases where the results of transcription of the transgene in tissues other than the target tissue would be deleterious.
Tandem repeats of two or more enhancer elements or combinations of enhancer elements may significantly increase transgene expression when compared to the use of a single copy of an enhancer element; hence enhancer elements find use in the
expression cassette. The use of two different enhancer elements from the same or different sources flanking or within a single promoter can in some cases produce transgene expression in each tissue in which each individual enhancer acting alone would
have an effect, thereby increasing the number of tissues in which transcription is obtained. In other cases, the presence of two different enhancer elements results in silencing of the enhancer effects. Evaluation of particular combinations of enhancer
elements for a particular desired effect or tissue of expression is within the level of skill in the art. Although generally it is not necessary to include an intron in the expression cassette, an intron comprising a 5' splice site (donor site) and a 3'
splice site (acceptor site) separated by a sufficient intervening sequence to produce high level, extended in vivo expression of a transgene administered iv or ip can optionally be included. Generally, an intervening sequence of about 100 bp produces
the desired expression pattern and/or level, but the size of the sequence can be varied as needed to achieve a desired result. The optional intron placed 5' to the coding sequence results in high level extended in vivo expression of a transgene
administered iv or ip but generally is not necessary to obtain expression. Optimally, the 5' intron specifically lacks cryptic splice sites which result in aberrantly spliced mRNA sequences. If used, the intron splice donor and splice acceptor sites,
arranged from 5' to 3' respectively, are placed between the transcription initiation site and the translational start codon as diagrammed in (1), below. ##STR1##
Alternatively, the intervening sequence may be placed 3' to the translational stop codon and the transcriptional terminator or inside the coding region. The intron can be a hybrid intron with an intervening sequence or an intron taken from a
genomic coding sequence. An intron 3' to the coding region, a 5' intron which is of less than 100 bp, or an intron which contains cryptic splice sites may under certain condition substantially reduce the level of transgene expression produced in vivo.
However, unexpectedly, a high level of in vivo expression of a transgene can be achieved using a vector that lacks an intron. Such vectors therefore are of particular interest for in vivo transfection.
In some cases, it may be desirable to use constructs that produce long term transgene expression in vivo, either by integration into host cell genomic DNA at high levels or by persistence of the transgene in the nucleus of cells in vivo in
stable, episomal form. Integration of the transgene into genomic DNA of host cells in vivo may be facilitated by administering the transgene in a linearized form (either the coding region alone, or the coding region together with 5' and 3' regulatory
sequences, but without any plasmid sequences present). Additionally, in some instances, it may be desirable to delete or inactivate a mutant gene and replace it with a desired transgene. This may be achieved by using an expression cassette suitable for
homologous recombination in vivo. Thus, for example, a linearized plasmid comprising an expression cassette may be used such as is described in European patent applications 88/201743.7 and PP89/202106.4. These applications disclose a plasmid for
targeting of a specific gene. For the present application, a linear plasmid can be constructed wherein the replacement gene is flanked by 5' and 3' sequences which are sufficiently homologous with the 5' and 3' sequences of the defective gene to provide
for homologous recombination. Where it desired to insert the replacement gene in the mutant gene (thereby inactivating it) a means for selection is included within the 5' and 3' flanking sequences of the plasmid.
The incidence of transgene integration into genomic DNA may be increased by incorporating a purified retroviral enzyme, such as the HIV-1 integrase enzyme, into the lipid carrier-DNA complex. Appropriate flanking sequences are placed at the 5'
and 3' ends of the transgene DNA. These flanking sequences have been shown to mediate integration of the HIV-1 DNA into host cell genomic DNA in the presence of HIV-1 integrase. Alternatively, the duration of the transgene expression in vivo can be
prolonged by the use of constructs that contain non-transforming sequences of a virus such as Epstein-Barr virus, sequences such as oriP and EBNA-1 which appear to be sufficient to allow heterologous DNA to be replicated as an episome in mammalian cells
(Buhans et al., Cell (1986) 52: 955).
Downstream from and under control of the transcriptional initiation regulatory regions is a multiple cloning site for insertion of a nucleic acid sequence of interest which will provide for one or more alterations of host genotype and modulation
of host phenotype. Conveniently, the multiple cloning site may be employed for a variety of nucleic acid sequences in an efficient manner. The nucleic acid sequence inserted in the cloning site may have any open reading frame encoding a polypeptide of
interest, for example, an enzyme, with the proviso that where the coding sequence encodes a polypeptide of interest, it should lack cruptic splice sites which can bleak production of appropriate mRNA molecules and/or produce aberrantly spliced or
abnormal mRNA molecules. The nucleic acid sequence may be DNA; it also may be a sequence complementary to a genomic sequence, where the genomic sequence may be one or more of an open reading frame, an intron, a non-coding leader sequence, or any other
sequence where the complementary sequence will inhibit transcription, messenger RNA processing, for example splicing, or translation.
A number of nucleic acid sequences are of interest for use in vivo gene therapy of lung diseases or diseases of other tissues. When it is desired to have an extra-pulmonary effect, nucleic acid providing for secretory leader sequence is included
in the expression cassette. Where the nucleic acid codes for a polypeptide, the polypeptide may be one which is active intracellularly, a transmembrane protein, or it may be a secreted protein. It may also code for a mutant protein for example are
which is normally secreted but which has been altered to act intracellularly. The nucleic acid may also be a DNA sequences coding for mRNA (antisense or ribozyme sequences such as those to HIV-REV or BCR-ABL sequences) or for proteins such as
transdominant negative mutants which specifically prevent the integration of HIV genes into the host cell genomic DNA, replication of HIV sequences, translation of HIV proteins, processing of HIV mRNA or virus packaging in human cells; the LDL (low
density lipoprotein) receptor, which specifically lowers serum cholesterol, and which can reduce the risk of heart attack in individuals with elevated serum cholesterol levels, and proteins such as granulocyte macrophage colony stimulating factor
(GM-CSF) which can stimulate the production of white blood cells from the bone marrow of immunocompromised patients and produce significant anti-tumor activity or cystic fibrosis transmembrance conductance regulator (CFTR) for treatment cystic fibrosis.
These, or other beneficial (therapeutic) nucleic acid sequences can be expressed in appropriate cells in vivo using this invention.
Examples of beneficial therapeutic nucleic acid sequences are those encoding molecules have superoxide dismutase activity or catalase activity to protect the lung from oxidant injury; endothelial prostaglandin synthase to produce prostacyclin and
prostaglandin E2; and antiprotease alpha-1 antitrypsin. Thus, this approach could dramatically improve the treatment of acquired immune deficiency syndrome (ADS), cystic fibrosis, cancer, heart dim, autoimmune diseases and a variety of life th | | |
