The new frontier: gene and oligonucleotide therapy

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Pharmaceutica Acta Helvetiae 68 (1994) 145-159

Review

The new frontier: gene and oligonucleotide therapy Hans Schreier Center for Lung Research, Vanderbilt University School of Medicine, B 1308 MCN, Nashville, TN 37232-2650, USA (Received 1 September 1993)

Abstract

Gene and oligonucleotide therapy are emerging as clinically viable therapeutic regimens for genetic, neoplastic, and infectious diseases. Approaches include insertion of human genes in viral vectors including recombinant retrovirus, adenovirus, adeno-associated virus, and herpes simplex virus-l, or recombinant bacterial plasmids. Viral vectors transfect cells directly; plasmid DNA is delivered with the help of cationic liposomes (lipofection), polylysine conjugates, gramicidin S, artificial viral envelopes or other such intracellular carriers. Major areas of interest include replacement of the cystic fibrosis transmembrane regulator gene and the ai-antitrypsin gene; arrest of human immunodeficiency virus infection; and reversal of tumorigenicity and cancer immunization, among others. Oligonucleotide therapy is principally focusing on the same areas, although the approach is to halt DNA transcription or messenger RNA translation with code-blocking triple-helix-forming or "antisense" oligomers. Contributions from the pharmaceutical sciences are expected in pharmaceutical chemistry, drug delivery systems design, analytical chemistry, and biopharmaceutics. Key words: Gene therapy; Gene marker; Gene therapeutics; Retroviral vector; Adenovirus vector; Adeno-associated virus vector; Herpes simplex virus-1 vector; Plasmid DNA; Cationic liposomes; Lipofection; Polylysine; Lipopolylysine; Gramicidin S; Artificial viral envelope

1. Introduction Abbreviations." AAV, adeno-associated virus; ADA, adenosine deaminase; AIDS, acquired immunodeficiency syndrome; AV, adenovirus; bGH, bovine growth hormone; CAT, chloramphenicol acetyl transferase; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane regulator; CMV, cytomegalovirus; DC-Chol, 3/3-[N-(N',N'-dimethylaminoethane)-carbamoyl]-cholesterol; DDAB, didodecyldimethylammonium bromide; DNA, desoxyribonucleic acid; DOPE, dioleoylphosphatidylethanolamine; DOSPA, 2,3-dioleyloxy-N[2(sperminecarboxamide)ethyl]-N,N-dimethyl-l-propanaminium trifluoroacetate; DOTAP, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium methylsulfonate; D O T M A , N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride; halaT, human a 1antitrypsin; hGH, human growth hormone; HIV-1, human immunodeficiency virus-l; HSV-1, herpes simplex virus-l; HTLV-III, human T cell lymphotropic virus type III; ICAM-1, intercellular adhesion molecule-l; IL, interleukin; lacZ, /3-galactosidase gene; LTR, long terminal repeat; MMTV, mouse mammary tumor virus; mRNA, messenger ribonucleic acid; RSV, Rous sarcoma virus; SCID, severe combined immunodeficiency; TIL, tumor-infiltrating lymphocytes; TNF, tumor necrosis factor; tPA, tissue plasminogen activator. 0031-6865/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI: 0 0 3 1 - 6 8 6 5 ( 9 3 ) E 0 0 1 8 - C

Over 4500 human diseases are classified as being of genetic origin (McKusick, 1988). Until now, substitution therapy, e.g., of enzyme storage diseases such as Gaucher's disease, and symptomatic treatment of gene deficiency-related organ malfunction and tissue deterioration, as in the case of cystic fibrosis, muscular dystrophy and others, have been the only means of treatment, while it has been a priori impossible to cure genetic diseases. Accordingly, the life expectancy of patients suffering from genetic defects is short, and, generally, their quality of life is severely curtailed due to both primary and secondary illness-related complications as well as the side effects of life-long medication. With the advent of gene manipulation by biotechnological techniques it became feasible to splice and insert human genes into viral or bacterial gene vectors.

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I~iral gene nfection) /

T "viral protein / newvirus~

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I defectgene missinggene

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Fig. 1. Progress in the level of rational treatment of genetic (incl. neoplastic) diseases and viral infections. As we become more apt in finding and defining defect or missing genes, or invading viral genes, respectively, we progress from the lowest level of alleviating clinical symptoms to the level of replacing or inhibiting proteins (thus intercepting the cascade of pathologic events), to the highest level of curing a genetic disease by either inhibiting its mRNA or replacing a defect or missing gene.

Hence, it was now principally feasible to use such recombinant material to replace a defect or missing gene, or insert a new gene into a human cell. Various routes of delivering genes ex vivo and in vivo have since been devised, as will be discussed here, and human gene therapy has become a clinical reality (Anderson, 1992). More recently, so-called "antisense" molecules (as opposed to the natural "sense" D N A or RNA) have become available, short synthetic nucleic acid sequences which align either with D N A (triple-helix formation) or with messenger R N A (mRNA) and arrest protein synthesis by inhibiting transcription or translation (Inouye, 1988; Miller and Ts'o, 1988; Stein and Cohen, 1988). With gene and oligonucleotide therapy as clinically feasible regimens, the rationale for therapy of a large number of genetic, viral and neoplastic diseases has changed dramatically. As shown in Fig. 1, until recently our approaches to the treatment of genetic defects were restricted to the lowest level, i.e., to alleviate the clinical symptoms, or, somewhat more rationally, to replace a missing protein. In contrast, gene and oligonucleotide therapy provide an opportunity to either replace the missing or defect gene at the origin (i.e., cure the disease in the true sense of the word) or

arrest undesired gene expression (specifically viral and oncogene expression) at the origin, i.e., at the DNA or m R N A level.

2. Gene therapy Today, we envision different modes of gene therapy for a variety of pathological manifestations which go beyond the original definition of gene therapy (Roemer and Friedmann, 1992).

2.1. Gene markers Genes have first been introduced into human cells ex vivo and re-injected into the host as "marker genes" to monitor the state of disease, and the duration of the modified cells in the diseased tissue. In order to follow the in vivo fate of tumor-infiltrating lymphocytes (TILs) (Rosenberg, 1991) or hepatocytes in the liver (Ledley et al., 1991), cells were transfected ex vivo with a gene construct encoding for the neomycin phosphotransferase gene which can be detected and monitored in tissue samples. 2.2. Gene replacement The original idea of gene therapy was to replace a defect or missing gene in a human cell to correct its malfunction. The most typical (and most extensively investigated) disease to benefit from such an approach is cystic fibrosis (CF), i.e., the replacement of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) gene which causes a permanent closure of chloride channels in epithelial cells resulting in the known clinical manifestations of CF (Berger et al., 1991; Hyde et al., 1993). Other typical examples in this category would be human al-anti-trypsin ( h a l a T ) (Kay et al., 1992), fl-glucocerebrosidase (Gaucher's disease) (Nolta et al., 1992), and adenosine deaminase (ADA) deficiency (Blaese and Anderson, 1990). 2.3. Gene therapeutics A novel adaptation of gene therapy is the transfection of cells with nonresident genes in order to accomplish in situ expression of a pharmacologically beneficial protein, or create a site for further therapeutic intervention. In this application, genes would, in a certain sense, act like "drugs", generating a product with a specific pharmacologic effect. This type of "gene therapeutics" includes: (i) generation of pharmacologically active products (proteins, immunotoxins), for instance of soluble CD4 to combat infection with the human immuno-

H. Schreier / Pharmaceutica Acta Helvetiae 68 (1994) 145-159

deficiency virus-1 (HIV-1), which binds avidly to the CD4 receptor through its surface glycoprotein (gpl20) (Morgan et al., 1990); or production of an HIV-regulated diphtheria toxin A chain which would result in cell suicide upon infection and viral reproduction (Harrison et al., 1992); or production of tumor necrosis factor (TNF) in TILs to boost their anticancer efficacy (Rosenberg, 1991); (ii) "production" of secreted hormones or enzymes, e.g., human factor IX (Yao and Kurachi, 1992), ADA (Lynch et al., 1992) or h a l a T (Garver et al., 1987) (this application is in part identical to "gene therapy", yet production of the desired protein is not restricted to the physiological site of production but could be any suitable cell population in any organ, e.g., hepatocytes producing halaT); (iii) insertion of an artificial genetic cell "defect" sensitive to pharmacologic agents which can then he exploited for selective cell kill; this principle has been demonstrated in vivo in malignant brain cells which became sensitive to ganciclovir following transfection with the herpes simplex virus-1 (HSV1) thymidine kinase gene (Takamiya et al., 1993).

3. Oligonucleotide (antisense) therapy

Just as desirable as it may be to correct a mutant gene or introduce a missing gene into a cell, in many cases it is as important to introduce a potent inhibitor of a gene which is expressed to the detriment of the host, as is the case in neoplastic, infectious, and also some inherited diseases (for review see Cohen, 1991). Various "antisense" strategies have been developed. "Antisense" compounds are essentially short synthetic nucleic acid segments that fit into certain complementary nucleic acid segments in human or nonhuman (viral, bacterial, parasitic) DNA or mRNA, thus blocking replication or transcription of DNA, or arresting translation of mRNA in ribosomes. The outcome in either case is prevention of expression of undesired protein. (Antisense strategies are, in addition, used scientifically to study the function of genes and the resulting effects on cell morphology and function). Three principal therapeutic approaches are being explored: (i) antisense oligonucleotides designed to bind to mRNA and block RNA processing or translation; a preferred site for antisense attachment is the so-called splice junction in pre-mRNA (nuclear mRNA), which inhibits formation of cytosolic mRNA and is therefore most powerful in inhibit-

147

ing protein expression. Using this approach, Stevenson and Iversen (1989) were able to inhibit translation of the human immunodeficiency virus (HIV) tat RNA. (ii) triple-helix-forming oligonucleotides which bind in a sequence-specific manner in the major groove of duplex DNA (including sequence-specific alkylation which results in permanent incapacitation of the target DNA) (Shaw et al., 1991); (iii) random sequences of oligonucleotides with high affinity for specific target molecules which bind to extracellular proteins and inhibit their (enzymatic) activity, e.g., a DNA aptamer which binds to and inhibits thrombin (Wang et al., 1993). The first two types of oligonucleotides are combined under the term "code blockers", whereas the term "aptamers" has been coined for the third type (for review see Riordan and Martin, 1991), To date, a large body of information on the efficacy of oligonucleotides in vitro has been generated, while information on in vivo activity remains scarce and inconclusive. Growth inhibition of viruses including human T cell lymphotropic virus type III (HTLV-III), HIV-1, Rous sarcoma virus (RSV), HSV-1, vesicular stomatitis virus (VSV), influenza virus, tick-borne encephalitis virus, hepatitis B virus, sendai virus, SV40 virus, and papilloma virus; oncogenes including c-myc, N-myc, and N-ras; cellular genes including multiple drug resistance, T cell and epidermal growth factor (EGF) receptor, prothymosin, fl-globin, myeloblastin, intercellular adhesion molecule-1 (ICAM-1), interleukin-2 (IL-2), IL-la, IL-lfl and others; and transfected reporter genes including chloramphenicol acetyl transferase (CAT), placental alkaline phosphatase driven by HIV Tat response element, and CAT driven by human papilloma virus E2 response element (reviewed by Bischofberger and Wagner (1992) and Crooke (1992), incl. references).

4. Design of DNA-containing viral vectors

Genes can principally be introduced by injection into a tissue without prior manipulation (Lin et al., 1990; Wolff et al., 1990). However, the probability is minute that such "naked" DNA will, by some serendipitous mechanism, enter the cell nucleus, insert into the cell genome, and express a desired protein. More likely, such DNA would be enzymatically degraded by extra- and intracellular nucleases. Therefore, DNA has been inserted either in viral vectors (for review see Miller, 1989) or in bacterial

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H. Schreier / Pharmaceutica Acta Helvetiae 68 (1994) 145-159

deficient (Danos and Mulligan, 1988). As shown in Fig. 2, the helper cell will generate complete virions encapsulating the retroviral vector. These recombinant virus particles are harvested and used for transfection of target cells.

RETROVIRUS

oao

I

pOI

I

env

RETROVIRALVECTOR humangene

HELPER

]l

CELL

~~ Hgag retroviral I P°lvector l ~

Il~ O/M ~B I N ~AN OT

R U V S I

Fig. 2. Recombination of retrovirus as retrovira] vector and packaging in helper cell to generate recombinant virus. The viral genome encoding for the viral proteins gag, pol, and era' are replaced with the human gene. The new retroviral vector is transfected into a helper cell which contains replication-deficient virus which can supply the missing viral proteins to form a new virion. Note that the env gene is split from the gag and pol genes in order to minimize potential production of replication-competent virus. Virus is continuously produced by the helper cell and can be harvested from tissue culture supernatant for infection of target cells.

plasmids which can be introduced into cells either via the viral, or via alternative endocytic a n d / o r fusogenic pathways (see discussion of routes of administration below). 4.1. Retroviral vectors Retroviruses are RNA viruses which replicate as DNA proviruses. The essential "tool" of the retrovirus is reverse transcriptase which, after infection, produces a double-stranded DNA of the provirus flanked by so-called "long terminal repeats" (LTRs)on each side which promote expression of the (natural or inserted synthetic) viral gene. A detailed description of the retroviral life cycle can be found by Varmus and Brown (1988). As shown in the general scheme in Fig. 2, the generation of retroviral vectors consists of two steps. First, the desired recombinant gene is inserted into an incomplete copy of the virus in place of its own gag, pol, and env protein-coding sequences. This synthetic retroviral vector consists then of the inserted gene and the LTRs which contain the expression and promoter sequences necessary for the expression of the new gene. Secondly, the modified sequence is introduced into so-called packaging or "helper" cell lines which contain viral sequences to form capsids (the env, pol, and gag sequences are present) but are replication

4.2. Adenovirus (AV) vectors The design of vectors from AV, a double-stranded DNA virus, and adenovirus-associated virus (AAV), a single-stranded DNA virus (see below), is somewhat different from retroviral vector design. The viral genome (or selected parts thereof), following ligation with the desired human gene, is ligated with plasmid either directly through complementary restriction sites, or in vivo through overlapping fragments. Recombinant AV is then produced in suitable cell lines, with or without the need for helper AV (Berkner, 1988). 4.3. Adeno-associated virus (AAV) vectors Similar to AV, AAV is manipulated to contain the gene of interest and then ligated into a bacterial plasmid which is transfected into a helper cell line for generation of intact recombinant virus. Ohi et al. (1990) constructed a chimeric bacterial plasmid containing AAV DNA and cDNA encoding for human /3-globin. Various human cell lines were infected with this plasmid and recombinant AAV was recovered. This chimeric AAV infected human hematopoietic cells, e.g., the human erythroleukemia cell line K 562, and expressed human /3-globin (Dixit et al., 1991). 4.4. Herpes simplex virus-1 (HSV-1) vectors Design and preparation of HSV-1 vectors is essentially identical to the process described for AAV (Geller and Breakefield, 1988).

5. Design of bacterial plasmid DNA The plasmid DNA transfection approach is fundamentally different from the viral vector approach. Bacterial plasmid is not "infective" per se and, therefore, needs to be shuttled into the cell by an intracellular carrier system (see below). Furthermore, as bacterial plasmid is naturally functioning episomally without integration in the host genome, there is no limitation as to the cell type that can be infected. Although, for the same reason, cells will not be stably transfected and repeated dosing will be necessary to maintain a desired therapeutic effect. As shown in Fig. 3, plasmid DNA contains the following essential components:

H. Schreier / Pharrnaceutica Acta Heluetiae 68 (1994) 145-159

i c ulticl°ning 1aT | AT t FTR cZ

iferase

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l

/

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yaoyo,,o # Fig. 3. General structure of plasmid DNA. The five major components responsible for expression of the desired human gene are: (a) the multicloning site where the foreign gene is inserted; (b) the (poly)A (polyadenylation) site which regulates gene transcription and determines the half-life time of expression; (c) an ampicillin or tetracycline resistance site needed for positive selection; (d) a strong promoter sequence for efficient gene expression; and (e) the E. coli origin of replication site to generate multiple copies of the plasmid after transfection.

149

Natural oligodeoxynucleotides have limited utility as they are efficiently and rapidly degraded by serum and cellular nucleases. Therefore, the phosphodiester backbone has been synthetically modified such that the resulting synthetic oligonucleotides become nuclease resistant. The two most widely investigated forms of synthetic oligonucleotides include phosphorothioates and methylphosphonate analogs (see Fig. 4). Phosphorothioates are anionic, whereas methylphosphonates are nonionic, more lipophilic and, therefore, cross cell membranes more efficiently than ionic derivatives. An increasing number of synthetic variations of oligonucleotides continues to be synthesized including other neutral analogues of phosphodiester, e.g., (methoxyethyl)phosphoramidate, formacetal and 5'-thioformacetal derivatives (Matteucci et al., 1991). Other derivatives include reactive groups such as photo-cross-

B

HO

O

I X--P=O (i) (ii)

(iii) (iv) (v)

the multicloning site, where the foreign gene is inserted; the polyadenylation site, which regulates gene transcription and the half-life time of gene expression; an ampicillin or tetracycline resistance site for positive selection; a strong promoter sequence for efficient gene expression; the E. coli origin of replication site to generate multiple copies of the plasmid after transfection.

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6. Design of oligonucleotides

Oligonucleotides are short synthetic strands of nucleic acids which bind to DNA, mRNA, or extracellular proteins in a complementary fashion. On statistical grounds, any sequence of 17 bases should appear only once in the human genome. Hence, in order to impart target specificity on oligonucleotides they need to be anywhere from 15 to 18 nucleotides in length (15- to 18-mers) (which results in an average molecular weight of around 5000).

/ O tt Fig. 4. Basic structure of chemically modified oligonucleotides. B = nucleic acid base; X = O: (natural) phosphodiester; X = S: phosphorothioate; X = CH3: methylphosphonate; X = N R 2 : phosphoramidate. A large number of chemical modifications has been introduced to generate photo-crosslinking, alkylating, "switchback" and other active derivatives (for a detailed review of the chemistry of oligonucleotides see Goodchild (1990)).

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H. Schreier / Pharmaceutica Acta Heh,etiae 68 (1994) 145-159

linking (Giovannangeli et al., 1992) or alkylating moieties (Shaw et al., 1991). Peptide nucleic acids, i.e., polyamide oligomers, have been designed which strand invade duplex DNA and displace a DNA strand (Hanvey et al., 1992). Another important new development is the synthesis of oligonucleotides with 3'-3' internucleotide junctions, so-called "switchback" oligonucleotides which form triple-helix DNA by binding to both strands of a target DNA simultaneously (Froehler et al., 1992) (for an extensive review of chemical structures and modifications thereof see Goodchild, 1990).

7. Gene carrier systems

The approaches of delivering genes include: (i) retroviruses; (ii) adenovirus (AV); (iii) adeno-associated virus (AAV); (iv) herpes simplex-1 virus (HSV-1); (v) cationic liposomes ("lipofection"); (vi) other miscellaneous approaches. 7.1. R e t r o v i r a l v e c t o r s

Retroviral vectors have been widely employed, specifically as carriers for marker genes which are inserted into the target cells ex vivo and then reinjected into the original host. TILs are a preferred target for the insertion of neomycin phosphotransferase which confers neomycin resistance to the transfected cell (Aebersold et al., 1990; Rosenberg et al., 1990). Biopsies are taken from the host at certain time intervals and the tissue screened for neomycin resistance which serves as indicator for the in vivo distribution and half-life time of TILs. Hepatocytes have been marked in the same fashion to follow the in vivo fate of a hepatocyte transplantation in patients with acute hepatic failure (Ledley et al., 1991). In a rather ingenious example of "drug targeting" TILs have been transfected with the TNF gene with the aim to generate high levels of TNF at the tumor site, thus preventing severe toxicity from systemic TNF levels (Rosenberg, 1991). The first human gene replacement therapy using a retroviral vector was approved for the replacement of the missing ADA gene in children suffering from severe combined immune deficiency (SCID) (Culver et al., 1991). Lymphocytes were harvested, transduced ex vivo with a retroviral vector containing the functional ADA gene, and reinfused into the host. Preliminary

data indicate significantly enhanced ADA levels and improved immune function in these patients. Hepatic genetic deficiencies have been experimentally treated with genes delivered via retroviral vectors. Chowdhury et al. (1991) reported reduced hypercholesterolemia in rabbits following ex vivo treatment and reimplantation of hepatocytes with the LDL receptor gene. Similarly, Soriano et al. (1992) transfected human hepatocytes with the neomycin resistance gene in a retroviral vector and transplanted the modified cells into the spleen of SCID mice. The efficiency of transduction of these human cells was found to be an order of magnitude lower (~ 1%) than the efficiency commonly found in rodent cells. A major focus of gene therapy is in the area of human immunodeficiency virus-1 (HIV-I) infection. As HIV-1 gp120 binds selectively and with high affinity to the CD4 receptor, it has been reasoned that a high concentration of circulating soluble CD4 could compete with cellular CD4 for HIV binding and thus intercept circulating HIV-1 virus before it can reinfect host cells. Hence, Morgan et al. (1990) proposed to transfect cells with the soluble CD4 gene and implant transformed cells into the infected host by direct infusion or cellular implants. Another such competitive strategy involves the introduction (and production) of mutant HIV-1 envelopes which would compete with the wild-type, occupying the CD4 receptor, and, therefore, inhibit the spread of the wild-type virus (Buchschacher et al., 1992). A third so-called "suicide" strategy involves transfection of T-lymphocytes with a HIV-regulated diphtheria toxin A chain gene. Expression of this gene initiated by HIV infection would cause cell death and, therefore, arrest spreading of the HIV infection (Harrison et al., 1992). A second major area of interest is cancer gene therapy. It has been shown in animals that cytokine genes introduced directly into tumor cells render these cells incapable of forming tumors, but also protect the animal against challenge with unmodified tumor, i.e., essentially immunize the animals against the tumor (Gansbacher et al., 1990; Golumbek et al., 1991). Another interesting application of gene therapy is in the cardiovascular area. It has been shown in the Yucatan minipig model that both endothelial cells (Nabel et al., 1989) and vascular smooth muscle cells (Plautz et al., 1991), when transfected ex vivo with the /3-galactosidase marker gene, could be inserted in predetermined arterial segments using an intravascular catheter with a proximal and distal balloon. Histochemical staining for /3-galactosidase expression was positive 2-4 weeks after treatment. Similarly, ADA has

H. Schreier / Pharmaceutica Acta Helvetiae 68 (1994) 145-159

been expressed in vascular smooth muscle ceils in the rat (Lynch et al., 1992). Nabel et al. (1990) demonstrated later using the balloon technique that retroviral gene vectors could be directly instilled into the arterial bed and found /3-galactosidase marker expression 21 weeks following gene transfer. Another unique application of gene therapy is the use of transfected endothelial cells as "coating" material for prosthetic devices (Wilson et al., 1989) with the aim to continuously release thrombolytic agents, e.g., tissue plasminogen activator (tPA) (Dichek et al., 1991). The use of retroviral vectors is extremely efficient and results in stable insertion of the transfected gene into the host genome. However, retroviral vectors have some significant limitations and potentially severe toxic manifestations which render their wider use in human gene therapy unlikely. While retroviruses infect cells fairly indiscriminately, they can only insert their genetic information into the host genome during the process of mitosis (Miller et al., 1990). Hence, an important restriction of using retroviral vectors is the fact that target cells must be replicating in order to be stably infected by the retroviral gene construct. Furthermore, if only one particular type of cells were to be transfected, e.g., transfection of marker genes into lymphocytes, this can currently only be performed ex vivo due to the promiscuous infectivity of retroviruses (one could speculate of attaching a "homing" device to the construct which would restrict its infectivity to the desired cell type, although such systems are currently not available). Another limitation of retroviral vectors is the fact that only about 8-kilobase pairs of foreign DNA can be encapsidated in the space which is normally occupied by the virus' own protein-coding sequences. A major concern of the retroviral approach is potential production of replication-competent viruses through insertion of the vector into the helper cell genome, leading to the restoration of the encapsidation sequence in the helper genome (Hu et al., 1987). However, strategies have been designed to essentially eliminate this problem, e.g., through the use of split viral genomes (coding sequences for core proteins and envelope protein are in separate genomes; see Fig. 2) and replacement of the downstream LTR with other polyadenylation sequences. Finally, the most serious concern with the use of retroviral vectors is the probability that, due to random genome insertion, insertional activation of protooncogenes can potentially happen which would lead to malignant transformation of the infected cell. After all, it must be kept in mind that retrovirus vectors are

151

laboratory analogues of highly oncogenic retroviruses. In one such event, three of a group of ten primates developed T-cell lymphoma following retroviral transfection (Donahue et al., 1992).

7.2. Adenovirus (AV) vectors AV is a double-stranded DNA virus. In contrast to retroviral vectors, AV can be loaded with up to 36-kilobase DNA segments and can be used for in vivo transfection as it also infects nonreplicating ceils. Upon cell entry via endocytosis and release into the cytoplasm, the AV gene remains episomal without insertion into the host genome (Horwitz, 1990) which eliminates the potential for insertional mutagenesis, the major obstacle with retroviral vectors. Recombinant AV vectors have been designed which are replication deficient (Berkner, 1988) and contain tissue-specific promoters which restrict the site of transgene expression (Friedman et al., 1986; Karlsson et al., 1986). While similarly broad in its range of infectivity as retroviruses, AV infects the pulmonary epithelium particularly avidly (Straus, 1984) and has indeed been most successfully employed in the in vivo transfection of pulmonary epithelial ceils with the h a l a T gene (Rosenfeld et al., 1991) and the CFTR gene (Rosenfeld et al., 1992), both in the cotton rat. Expression of h a l a T persisted for 1 week, whereas the CFTR protein was detected for up to 2 weeks, and CFTR transcripts for up to 6 weeks following transfection. AV gene constructs have also been employed to correct ornithine deficiency in vivo by transfecting the gene for ornithine transcarbamylase in a mouse model (Stratford-Perricaudet et al., 1990), and to demonstrate lacZ gene expression (/3-galactosidase activity) in skeletal and cardiac muscle following intravenous and intramuscular administration in mice (Stratford-Perricaudet et al., 1992). As with all viral approaches, of major concern with AV vectors is recombination with wild-type AV, specifically in the upper airways, which would lead to the generation of replicating AV with unknown pathogenicity. Due to the ubiquity of AV infection, a significant fraction of the population has developed immunity to AV (Straus, 1984) which would greatly reduce the efficiency of transfection. Development of immunity during the course of treatment is likely as the AV vector remains episomal and requires repetitive dosing. Whether repeated exposure of the pulmonary epithelium to recombinant AV would create any acute or chronic tissue or cell toxicity other than immunity is unknown at this point.

H. Schreier / Pharmaceutica Acta Heh'etiae 68 (1994) 145-159

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approach. However, some potential disadvantages and toxic side effects of AAV are of concern. While no disease has been associated with AAV infection, its in vivo effect on target cells is unclear. It has been shown that cell growth may be untowardly affected by AAV infection (Winocour et al., 1988).

7.3. Adenovirus-associated (AA V) viral vectors AAV is a parvovirus which contains a single strand of DNA of 4.7 kb. Human AAV infection appears to be nonpathogenic with a majority of the population testing positive for AAV capsid protein antibodies (Parks et al., 1970). Of great interest for gene therapy is the preferential site-specific insertion of wild-type AAV in chromosome 19 (Samulski et al., 1991), while integration of recombinant replication-deficient AAV has also been mapped in chromosomes 21 and 22 (Muzyczka, 1992). AAV vectors have been used to transfect a human leukemia cell line (Dixit et al., 1991) (with the goal of eventually transfecting thalassemia and sickle-cell erythrocytes) and a cystic fibrosis cell line (Flotte et al., 1992), and have also been applied successfully in vivo to transfect a lung lobe with CAT in Sprague-Dawley rats (Flotte et al., 1993). At this point, the experimental data base available is too small to definitely assess the advantages of this

7.4. Herpes simplex virus-1 (HSV-1) vector HSV-1 can, like AV, infect nondividing cells and has potentially the largest DNA carrying capacity of all viral vectors. It has gained particular interest as a means to infect neurons (Geller and Breakefield, 1988). However, Johnson et al. (1992) have shown that replication-deficient HSV-1 vector apparently continues to be cytopathic. A detailed description of viral vectors, their advantages and disadvantages, as well as their potential applications is provided in a recent review by Roemer and Friedmann (1992).

Table 1 Frequently used cationic lipids which form cationic liposomes with dioleylphosphatidylethanolamine (DOPE) in various molar ratios. DNA is added in variable ratios to form cationic liposome DNA complexes Chemical n a m e / S h o r t name/Commercial name N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium a chloride/DOTMA/Lipofectin T M methylsulfonate/DOTAP b

~

O

~ . . ~ N(CH3)3 H'"dH

didodecyldimethylammonium bromide C/DDAB/LipofectaceXm

A/Vk/kA/

+/ N.~

2,3-dioleyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate d/ D O S P A /LipofectaminXM

~

o

~

cn3 O

O N

CH 3

NH z N H

3

NH

+ NH s 3fl-[N-(N ',N '-dimethylaminoethane)-carbamoyl]-cholesterol e/DC-Chol

CH 3 References: a Feigner et al., 1987; b Stamatatos et al., 1988; c Pinnaduwage et al., 1989; d Hawley-Nelson et al., 1993; ~ Gao and Huang, 1991.

H. Schreier / Pharmaceutica A cta Helvetiae 68 (1994) 145-159 7.5. Cationic liposomes

A novel and highly efficient DNA transfection technique called "lipofection" was introduced by Feigner et al. in 1987. The lipofection reagent consists of the positively charged quaternary amino lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) in a 1:1 weight mixture with dioleylphosphatidylcholine (DOPE) which is now commercially available as Lipofectin®. Several alternative positively charged lipids for gene transfection, shown in Table 1, have since been introduced. This class of lipid-based transfection agents has been employed successfully for a variety of cell lines in vitro and has recently also been shown to be active in vivo. Luciferase mRNA was transfected into NIH 3T3 cells using the D O T M A - D O P E liposome complex (Malone et al., 1989). Brigham et al. (1989a) reported expression of the CAT gene inserted into a RSV plasmid (pRSVCAT) in bovine pulmonary artery endothelial cells using "lipofection". Debs et al. (1992) achieved expression of CAT in rat lung alveolar type II cells, alveolar macrophages, and several human lung carcinoma cell lines following transfection with D O T M A / D O P E - D N A complexes, with CAT activity persisting up to 35 days. Brigham et al. (1989b) were the first to successfully use Lipofectin® in vivo. The CAT reporter gene driven by a SV40 promoter (pSV2CAT) was expressed preferentially in the lungs of mice when given by either the intravenous or the intratracheal route. More recently, expression of h a l a T in rabbit and sheep lung was demonstrated following the same protocol (Canonico et al., 1992). Hazinski et al. (1991) showed similarly expression of CAT in rat lung following instillation of two CAT plasmids, driven by a RSV promoter or a mouse mammary tumor virus (MMTV) promoter. As the latter promoter sequence contains glucocorticoid response elements, dexamethasone treatment of pMMTVCAT-instilled animals resulted in upregulation of CAT expression. Similarly, Stribling et al. (1992) aerosolized mice with CAT expression plasmid and found high levels of CAT activity in the lung, both the airway epithelium and the alveolar lining. Other in vivo applications of this technology include transfection of selected segments of arterial endothelial cells and systemic vessels via balloon catheterization (Nabel et al., 1990; Lim et al., 1991). Intraperitoneal administration of cationic liposome CAT complexes resulted in efficient transfection of T lymphocytes in mice (Philip et al., 1993). Generalized CAT gene expression throughout the body was reported following intravenous injection of a formulation con-

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taining pCMVCAT complexed with D O T M A : D O P E at ratio of 1/zg DNA : 8 nmol (4.8/zg) lipid over a dose range of 50 to 150/zg in mice (Zhu et al., 1993). Recently, Hyde et al. (1993) have for the first time demonstrated correction of the ion transport defect in cystic fibrosis transgenic mice by delivering a CFTR expression plasmid complexed with Lipofectin® to the lungs of these mice via instillation. The use of cationic liposomes as opposed to viral vectors for the gene delivery has a number of technological as well as therapeutic and toxicological advantages: (i) the complexation of cationic lipid and DNA is essentially quantitative, hence no separation of unencapsulated DNA from the complex is necessary; (ii) the production of cationic lipid-DNA complexes is a rapid, highly reproducible and simple one-step procedure; (iii) plasmid DNA vectors are directly complexed with the cationic lipids without the need for virus production and screening; (iv) as essentially all biological surfaces carry a net negative charge, interaction of cells with cationic liposome-DNA complexes and transfection is spontaneous; yet, to date only very low efficiencies of delivery can be achieved due to currently unknown reasons; (v) the DOPE component of cationic liposomes is believed to be responsible for the escape of the complex (or DNA?) from the endosomal compartment into the cytosol, an essential step for the successful delivery of DNA to the cell nucleus; (vi) cationic liposomes are per se nonimmunogenic, hence repeated dosing will most likely not result in antibody neutralization or untoward immune response. Open questions and skepticism remain concerning the safety of cationic liposome-DNA complexes although safety data have been generated on a limited scale. Not unexpectedly, positively charged liposomes (in the absence of DNA) interact spontaneously and indiscriminately with plasma components, red blood cells and cell membranes of the vasculature upon intravenous administration. Plasma turbidity, hemolysis and clot formation have been observed in vitro (Senior et al., 1991). However, recent in vivo safety studies of cationic liposome-DNA complexes have generated encouraging results. Lung function and histology in rabbits were found unchanged following weekly doses of 2.5 mg

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L i p o f e c t i n ® / D N A complexes given either by aerosol or by intravenous injection for 4 weeks (Plitman et al., 1992). Another recent report found that intravenous or intra-tumor injection of DNA-cationic liposome complexes had no adverse effect on histology, serum enzymes, creatinine, or cardiac function in mice (Stewart et al., 1992). More systematic studies of the cellular interaction and related cell toxicity of various cationic lipid compounds are only emerging at this point. Farhood et al. (1992) reported a correlation between the protein kinase C inhibitory activity of cationic cholesterol derivatives carrying a quaternary amino group vs. those with a tertiary amine function. As protein kinase C may play a role in gene expression, monitoring its activity as a function of exposure of cells to cationic liposomes may be an indicator for the potential in vivo toxicity as well as transfection efficacy of the carrier. Gene delivery by means of l i p o s o m e - D N A complexes (including other non-cationic liposome formulations such as conventional negatively charged liposomes, virosomes, pH-sensitive liposomes, etc.) has been reviewed by Mannino and Gould-Fogerite (1988), Nicolau and Cudd (1989), Felgner and Rhodes (1991), Hug and Sleight (1991), Litzinger and Huang (1992), and Brigham and Schreier (1993). A recent issue of the Journal of Liposome Research (Vol. 3, No. 1, 1993) is entirely dedicated to the topic of cationic liposomes.

7.6. Miscellaneous alternatiue approaches Various investigators have shown delivery of DNA by endocytic pathways using poly-L-lysine as the cationic carrier. In order to impart tissue selectivity, the carrier was covalently coupled to a receptor-selective targeting molecule such as asialoorosomucoid, a galactose-terminal asialoglycoprotein targeted to the hepatocyte asialoglycoprotein receptor (Wu and Wu, 1988; Wu et al., 1991), to human transferrin (Wagner et al., 1990; Curiel et al., 1992a), AV (Curiel et al., 1992b), or a n t i - t h r o m b o m o d u l i n antibody 34A (Trubetskoy et al., 1992). A synthetic lipophilic polylysine-phosphatidylethanolamine conjugate ("lipopolylysine") has also been used successfully as transfecting agent (Zhou et al., 1991; Zhou and Huang, 1992). Still another approach was employed by Huckett et al. (1990) who used insulin as receptor-specific carrier, linked to positively charged N-acylurea albumin, to deliver DNA to HepG2 cells by endocytosis via the insulin receptor. Wu and Wu (1988) demonstrated liver targeting of a CAT marker plasmid packaged with a polylysine-asialoorosomucoid conjugate. Following the same regimen,

they demonstrated partial correction of genetic analbuminemia in Nagase rats by hepatic transfection of the structural human albumin gene which resulted in albumin secretion (Wu et al., 1991). The ubiquitous transferrin receptor may serve as a target for more generalized gene delivery using a polylysine-transferrin conjugate (Wagner et al., 1990; Curiel et al., 1992a). A unique targeting effect to neoplastic cells may be achievable with this system due to the fact that rapidly dividing cells require a high level of iron, yet exhibit upregulated transferrin receptor density. An argument in favor of such alternative systems is the fact that delivery is accomplished through the physiologic pathway of endocytosis - in contrast to the perceived "forced" cell entry by cationic liposomes (although the predominant mode of entry of these systems may be endocytosis as well) - hence, toxicity should be at a minimum and repeated or prolonged treatment should be feasible. The disadvantage of this pathway, however, is the fact that the complex enters the endolysosomal pathway leading to enzymatic degradation of a majority of the delivered material (i.e., a very low "intracellular bioavailability"). Accordingly, augmentation of gene expression was accomplished using chloroquine to inhibit lysosomal degradation (Curiel et al., 1992a), although such manipulations are not feasible in vivo. In order to build a lysosomal escape mechanism into their polylysine-DNA complex, Curiel et al. (1992b) designed a hybrid compound consisting of the polylys i n e - D N A complex and AV, where the AV's function is to provide an escape mechanism from the endolysosomal compartment. While this approach, in principle, reintroduces the disadvantages of viral systems as discussed above, it also would have the advantage of not being limited by the DNA size, as the DNA is essentially packaged on the outside of the virus, rather than in its limited payload compartment. A similar hybrid system has recently been designed by Trubetskoy et al. (1992) who combined cationic liposomes consisting of the cationic 3/3-[N-(N ',N 'd i m e t h y l a m i n o e t h a n e ) - c a r b a m o y l ] - c h o l e s t e r o l and D O P E ( D C - C h o l / D O P E ) and a CAT marker genecarrying N-terminal modified poly(L-lysine)-antibody conjugate directed to mouse lung endothelial cells. A ternary electrostatic complex with the anti-thrombomodulin antibody 34A as the targeting device exhibited a 10- to 20-fold enhanced transfection efficiency in mouse lung endothelial cells in culture when cationic liposomes were present. A synthetic lipophilic derivative of polylysine,

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poly(L-lysine)-phosphatidylethanolamine (lipopolylysine), has been designed by Zhou et al. (1991) and has been employed in combination with DOPE for gene transfer in vitro. It has recently been shown by electron microscopy that this complex enters cells in coated pits via an endocytic pathway and that DOPE plays an integral role in the release of the DNA from the endosome (Zhou and Huang, 1992). Similarly, Debs et al. (1992) successfully employed lysinyl phosphatidylethanolamine- and cholesteryl-flalanine derivatives for DNA transfection of a variety of cell lines. Another alternative protocol using a mixture of the cyclic cationic amphipathic peptide gramicidine S and DOPE has been introduced by Legendre and Szoka (1993). Comparing Lipofectin® and their gramicidin S - D O P E - D N A complex, they reported a 2- to 20-fold enhancement of transfection of the luciferase gene into nonphagocytic cell lines including CV-1, HeLa and HepG2 cells, with their own construct. Finally, Chander and Schreier (1992) designed an artificial viral envelope exhibiting HIV gpl20, which binds avidly and selectively to CD4 receptor-positive cells such as T lymphocytes. CAT expression in CD4 + cells was demonstrated when such gpl20 artificial viral envelopes were employed (Schreier et al., 1992).

8. Oligonucleotide carrier systems

Similar to plasmid DNA, the major problem of oligonucleotide delivery is their limited access to the intracellular (and intranuclear) space. Chemical means to overcome cell exclusion, e.g., the design of more lipophilic compounds, are limited as they may compromise the selectivity and binding affinity for the intended target DNA or RNA. Hence, suitable intracellular carrier systems, similar as the ones employed for gene delivery would be the best solution to overcome this crucial obstacle. The use of cationic liposomes to deliver antisense constructs is only emerging at this point. Both cellular as well as intranuclear delivery of an antisense oligonucleotide which hybridized to ICAM-1 was found increased by up to three orders of magnitude in the presence of a cationic liposome carrier (Lipofectin®). Interestingly, while the (fluorescence-labeled) oligonucleotide appeared to remain within cytosolic, most likely, endosomal compartments in the absence of the cationic lipid carrier, in its presence, the molecule was, by some as yet unknown mechanism, released from the endosomal vacuole and efficiently shuttled to the cell

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nucleus (Bennett et al., 1992, 1993). In vivo data on such regimens are not available yet.

9. Conclusions and outlook

Clearly, gene and oligonucleotide therapy is a revolutionary approach: on a large scale, we have now the opportunity to fight the cause of a disease rather than its symptoms. Accordingly, progress is swift. A Science article commemorating two (!) years of gene therapy in October 1992 (Thompson, 1992) listed 18 human gene therapy trials in progress, 11 of which were cancer, 2 AIDS, and 5 enzyme deficiencies (3 ADA) trials. The latest report in H u m a n Gene Therapy from June 1993 (Human Gene Marker/Therapy Clinical Protocols, 1993) lists 42 U.S. and 5 international (1 Chinese, 1 French, 1 Italian, 2 Dutch) clinical trials underway. While some of the strategies described here may still appear like science fiction to some, it should be kept in mind that we now, perhaps for the first time, have the distinct advantage of not only knowing the molecular target in great detail, but also of being able to predict and rapidly generate molecular structures (in a "sense" or "antisense" way) that will alleviate a defect or impart a new desired function in a cell. Hence, molecular drug design in this field is progressing with increasing momentum as a genuine and rather powerful interdisciplinary effort between molecular biology, cell biology, and classical pharmaceutical chemistry. Finally, the strongest impetus of all may be the fact that these new therapies are aimed at essentially fatal diseases. Patients dying from leukemias, AIDS, CF, and a variety of other neoplastic, infectious and genetic disorders are in desperate need for novel therapeutic strategies of which gene and oligonucleotide therapy appear to be the most promising yet. What may still hamper progress is our rather slow progress in designing and developing suitable drug carrier systems which protect the active agent, efficiently deliver it intracellularly, and, most importantly, are targetable to selective tissues or cell populations. Hence, from a pharmaceutical point of view, these "drugs" provide again (after the most recent biotechnology revolution has entered mainstream) a major challenge for the pharmaceutical scientist, mainly the pharmaceutical chemist, but also and in particular for the drug delivery systems designer whose task is to device intracellular, preferably targetable and nontoxic carrier systems for parenteral as well as alternative nonparenteral routes of administration. In addition, the development of suitable in vitro and in vivo analyti-

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cal, stability-indicating, pharmacokinetic, toxicologic and pharmacodynamic methodology will be prerequisite for the rapid "translation" of this emerging technology into clinically viable and therapeutically beneficial regimens. It should be mentioned that gene therapy has created its own unique set of ethical as well as environmental concerns which have been, and will continue to be addressed by the national and international regulatory bodies. A discussion thereof would, however, be beyond the scope of this review.

Acknowledgements I would like to thank my colleagues at the Center for Lung Research at the Vanderbilt University School of Medicine, Drs. A.E. Canonico and K.L. Brigham for providing a preprint of their manuscript "In Vivo Expression of Human Genes", and Dr. J.T. Conory for help with the DNA plasmid section. Financial support was provided by Advanced Therapies, Inc.

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