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Chapter 20 DNA- and Adenovirus-Mediated Gene Transfer into Cardiac Muscle
CHAPTER 20
DNA- and Adenovirus-Mediated Gene Transfer into Cardiac Muscle Alyson Kass-Eisler* and Leslie A. Leinwandt 'Cold Spring Harbor Laboratory Cold Spring Harbor, New York 11724 +Department of Molecular, Cellular, and Developmental Biology University of Colorado at Boulder Boulder. Colorado 80309
I. Introduction 11. Methods for Cardiac Injection 111. Homogenization of Tissue and Assays IV. Experimental Design V. Future Directions References
I. Introduction In this chapter we will compare two methods of gene transfer into the rodent heart: direct plasmid DNA injection and injection of recombinant adenovirus. The goals of cardiac gene transfer that will be considered here are defining the elements regulating cardiac gene expression and phenotypic modification. Most studies aimed at understanding cardiac gene regulation have used transient transfection of DNA constructs into fetal or neonatal cardiocyte cultures. No transfections of adult cardiocytes have been reported. However, these studies are limited because of the developmental stage difference between the cultured cells and the adult and because of the inability to reproduce complex physiological and pathological processes in a tissue-culture environment. One solution to this problem is the creation of transgenic mice bearing reporter genes whose expression is driven by various promoters and putative regulatory regions. However, the creation of transgenic animals for this purpose is both time-consuming and expenMETHODS IN CELL BIOLOGY. VOL. 52 Copyright Q 1998 by Academic Press. AU rights of reproduction in any form resewed. lN91-67YX/98 125.00
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sive. Direct gene injection using naked plasmid DNA was first described in skeletal muscle by J. Wolff et al. in 1990. Several laboratories have extended the use of this methodology to include heart, which appears to be much more efficient than skeletal muscle in taking up and/or expressing naked DNA (Lin et al., 1990; Ascadi et al., 1991; Buttrick et al., 1992). The predominant use of direct injection into the heart has been to analyze promoter elements in vivo using reporter genes driven by the promoter element of interest (Kitsis et al., 1991; Buttrick et al., 1993). There are several advantageous features of naked DNA-mediated gene transfer. Primary among these are the stable expression resulting from gene transfer (Wolff et al., 1992) and the relative simplicity of the approach, which avoids infectious agents. Reports of gene expression beyond 1 year exist (Wolff et al., 1992). In addition, the DNA remains episomal, avoiding potential risks associated with the integration of DNA into the host genome (Wolff et al., 1992). The major drawback to this approach is its relative inefficiency.The maximum activity that can be obtained from gene expression appears to be restricted to an area immediately surrounding the injection site (Buttrick et al., 1992). Furthermore, the number of cells that take up and express the DNA is relatively small, corresponding to -0.02% of the myocytes in the heart (Kitsis et al., 1993). These drawbacks do not affect the utility of this method for mapping the regulatory elements of genes. Substantial reporter gene activity is obtained even after the introduction of relatively small amounts of DNA. In fact, when contemplating gene regulation studies, it is important to use relatively small quantities of DNA if the promoter is very active. For example, a strong promoter such as the Rous sarcoma virus LTR is out of the linear range of dose responsiveness above -2 pg in a rat heart. The assumption is that there are limiting transcription factors and that the cell rapidly becomes saturated with DNA sequences that bind these factors. Therefore, a DNA dose-response curve should be generated for each promoter under investigation. The second general use of in vivo gene transfer is to examine the consequences of expression of a given gene on the structure or function of the heart. This could involve expression of a mutant protein or ectopic expression of an isoform of a protein normally found in another setting. For these purposes, unless the gene encodes a secreted protein, DNA injection would not be efficient enough for most forms of phenotypic modification. We have therefore turned to the use of recombinant adenoviruses. Adenoviruses are double-stranded linear DNA viruses that have been extensively characterized in clinical and laboratory settings. Adenoviruses efficiently infect a large number of replicating and nonreplicating cell types. Their ability to infect nonreplicating cells makes them ideal for use in the heart, since adult cardiac myocytes are terminally differentiated. The recombinant adenoviruses in current use by most laboratories are replication deficient because they are deleted in a major regulatory region, termed Ela. Vectors currently exist that can accommodate up to 8 kb of a recombinant sequence, which makes them potentially useful for the majority of cDNA se-
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quences (Bett et af., 1994). These viruses can also be produced as extremely high-titer stocks (1011-1012pfdml). Finally, adenoviruseshave an excellent safety profile. The genome rarely integrates into the host genome and has never been associated with human malignancy. Several groups have demonstrated very efficient infection of the cardiovascular system by recombinant adenoviruses (Kass-Eisler et af., 1993; Stratford Perricaudet et al., 1992). Our group has focused on direct intracardiacmuscle injection of viruses. We first determined that both fetal and adult cardiac myocytes can be efficiently infected in culture (Kass-Eisler et af., 1993), achieving virtually 100% infection with a multiplicity of infection (MOI) of 2-3 pfdcell. We then examined the amount and distribution of gene expression resulting from injection of various viral doses into the left ventricular wall of the rat heart. Between 6 X lo6 and 2 X lo9 pfu there is a linear response between viral dose and expression. We have also compared the efficiency of gene transfer of a plasmid and an adenovirus bearing identical transgenes. For these experiments, we have used an adenovirus of the serotype 5 that bears the CAT reporter gene driven by the cytomegaloviruspromoter. The CAT gene was chosen because of its ability to be quantitated as well as its ability to be detected immunohistochemically.At maximal dose responsiveness for virus and plasmid DNA, the amount of CAT activity was approximately lOOOX higher in hearts injected with virus, 5 days following injection (Kass-Eisler et af., 1993), compared with its level in hearts injected with plasmid DNA. We had previously demonstrated in hearts injected with DNA that 98% of the activity of reporter genes was restricted to a small area around the injection site and that the transfection efficiency was low, corresponding to only -2000 cells in the entire heart (Kitsis et al., 1993). We compared both the distribution and the proportion of CAT-positive cells resulting from a single virus injection with results obtained from DNA injection. The quantitative distribution of expression was no different between adenovirus and plasmid DNA injection, in that the majority of expression was restricted to the injection site. However, because the amount of gene expression was so much higher in the virus injections, there was significant gene expression throughout the entire heart. In order to determine the proportion of cells in the heart which were infected with the virus, sections of the heart were stained with an anti-CAT antibody. In the regions of the heart surrounding the injection site, virtually 100% of cells were expressing CAT. In contrast to naked DNA injection, which transfects only cardiac myocytes, all cell types in the heart were able to be infected by the virus. The proportion of CAT-positive cells decreased as a function of distance from the injection site, reaching -5-10% of the cells at the base of the heart, which was the most distal location relative to the injection site. An additional feature of adenovirus infections in the adult heart that must be taken into consideration is the transient expression of genes. A universal observation with adenovirus is that independently of the route of the virus or the transgene it expresses, expression is transient, dropping sharply within -2 weeks, reaching undetectable levels
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by 80 days (Kass-Eisler et al., 1994; Mittal et al., 1993; Quantin et al., 1991). It now seems clear that the long-term expression of genes is limited by the immune response of the host to the virus and to the transgene. This immune response appears to be both humoral and cellular (Kass-Eisler et al., 1994; Yang et al., 1994). Extremely high levels of neutralizing antibodies develop within 5 days following virus administration (Kass-Eisler et al., 1994). Antibodies directed against the transgene also develop, but with a temporal delay compared with those directed against the virus (Kass-Eisler et aL, 1994).Traditionalimmunosuppression by agents such as cyclosporine is ineffective in prolonging gene expression (Kass-Eisler et al., 1994; Engelhardt et al., 1994). Experiments are underway to examine the time course of gene expression in transgenic animals whose immune systems have been modified in a variety of ways. The response of the immune system could be potentially circumvented by introducing virus into the immunologicallyincompetent neonatal animal. Neonatal rodents are immunologically incompetent and therefore do not recognize viral or transgene proteins as foreign. Expression by adenovirus-mediated genes in a neonate appears to be indefinite, reaching a time course of at least 7-8 months (Kass-Eisler et al., 1994; Rosenfeld et al., 1991). Experiments are currently in progress to determine whether neonates can be tolerized to the virus, allowing multiple administration of various recombinant viruses. In the case of injection into neonates, the technique will be described later. Because of the size of the animals, direct cardiac injection is not feasible. Therefore, animals are injected percutaneously into the thoracic cavity. This mode of introducing virus results in effective infection of multiple organs, including heart, diaphragm, lung, and liver, for an indefinite period of time. The amount of CAT activityper heart is approximately 1OOOX lower than that seen from direct cardiac injection. The lower levels of expression in the heart are most likely due to the distribution of virus by this route of administration. Thoracic-cavity injection most likely results in the majority of adenovirus being introduced into the lung. In support of this, 100-fold greater expression levels can be measured in the lungs of neonatally injected animals than in the lungs of directly cardiac-injected adults (Kass-Eisler et al., 1994).
II. Methods for Cardiac Injection Our injection protocol involves a simple surgical procedure to exteriorize the heart followed by injection of the heart muscle with a hypodermic needle and is usually performed by two people. Although other injection protocols have been developed for various species (Hansen et aZ., 1991; von Harsdorf et aL, 1993), the following protocol is for adult rats. This procedure can be used for delivery of naked DNA as well as recombinant adenoviruses. Animals are anesthetized with an intraperitoneal injection of 4% chloral hydrate (made fresh with water) at a dosage 0.75 d l 0 0 g body weight. This
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anesthesia, which takes effect within -10 min, lasts for approximately 30 min of deep sleep followed by approximately 1-2 hr of partial anesthesia. When animals are properly anesthetized there should be a loss of corneal reflexes and a lack of withdrawal to painful stimulation. Once anesthetized the animal is laid in a supine position. A vertical incision is made slightly to the left of midline with either a scalpel or sharp scissors. This can be accomplished through a natural slit in the muscles that runs diagonally across the animal’s left chest. The chest muscles are then blunt-dissected to the level of the ribs. The apical impulse of the heart can then be visualized (avoid the pulsation coming from the nearby great vessels). The rib just below the apical impulse is then cut with a scissors 5 mm to the left of the sternum. An incision too close to the sternum would risk damaging the internal mammary artery. The scissorsshould be held perpendicular and inserted as shallowly as possible to avoid damage to the heart or lungs. A Crile forceps is then inserted into the chest cavity through the rib incision perpendicular to the chest wall. The incision is then gently widened with the opening of the forceps. This opening of the chest cavity will cause the lungs to deflate, preventing respiration; therefore, it is important to continue as quickly as possible. The heart can usually be seen in the chest cavity at this point. If not, gentle pressure the right side of the chest can help move the heart into view. The heart is then gently exteriorized by grasping its apex with the Crile forceps. It is very important not to grip the heart by the atria or the great vessels, or to grasp the lungs. The heart can then be gently held by the operator’s fingers while the second operator performs the injection. A 100-pl Hamilton syringe with a 27-gauge needle containing the injectate is then inserted into the wall of the left ventricle. We have generally used an injectate volume of 50 p1 PBS (137 mM NaCl; 2.7 mM KCl; 10 mM Na2HP04; 2 mM KH2P04,pH 7.4)’ but this volume can be varied from 10 to 100 pl (Wolff et al., 1991) if necessary for a rat heart. The heart is then gently placed back into the chest cavity. The chest of the animal is then gently squeezed with fingers to expel any air from the cavity. The lungs are inflated using a Harvard small-animal respirator with a nose-cone adapter that fits over the snout of the rat. The nose cone is easily constructed by cutting of the bottom 4 cm of a 30-ml syringe and attaching a piece of glove latex over the wide end. The cone is attached to the respirator through tubing placed on the Luer-lock end of the syringe. The need for intubating the trachea is eliminated with the use of the nose cone, which provides positive-pressure ventilation of the animal. Two to four respirations with the respirator should be enough to induce spontaneous respiration; additional use should be kept to a minimum. At this point the muscle layers of the chest are closed using 3-0 silk sutures. Complete evacuation of pneumothorax should be accomplished before final suturing of the chest by gentle squeezing of the chest. The skin can then be stapled closed. Approximately 10%of all animals will succumb during this surgical
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procedure. The two main causes of death are improper anesthetic dosage and the trapping of air in the chest. Unlike rats, mice do not survive this procedure as well without modification. We have recently injected mice with adenovirus directly into the thoracic cavity. To do this, mice are held gently between the thumb and forefinger, and a 26gauge needle containing virus in a volume of 30 pl is introduced into the chest near the site of the heart. Care must be taken to insert and remove the needle slowly and gently. Complete immobilization of the animal is essential. Approximately 10% mortality (usually immediately after injection) can be seen with this procedure. Thoracic-cavity injection is also quite effective in the infection of neonatal animals. Neonatal animals are quite transparent, and if they are held over a light source, the ability to inject near the heart is greatly enhanced. Mortality rates from the injection in neonates are only about 2%.
III. Homogenization of Tissue and Assays For CAT (chloramphenicol acetyl transferase) and luciferase assays, tissues are homogenized. We have also used /3-galactosidase as a reporter gene, in which case whole tissues are harvested for staining. In addition, we have used human growth hormone (Selden et aZ., 1986) as a reporter gene in rats. Although high levels of expression of hGH can be measured in heart following administration of either phGH or AdhGH by direct cardiac injection,there is very little secretion of the hormone from the heart (see Fig. l),although detectable amounts in serum allow for multiple measurements on the same animal over time. In addition, hGH activity can be measured in homogenized tissue as discussed later. Before sacrifice, animals are given a large dose of 4% chloral hydrate (-1.5 ml/lOO g body weight). This is potentially a lethal dose, so care should be taken not to let the animals expire before the removal of tissues. The heart is removed by an abdominal incision through the midline. The ribs on both sides of the xiphoid process of the sternum are cut, revealing the heart in the chest cavity. The heart is then held with forceps by the apex while being cut at the base of the heart with a pair of scissors. The heart is immediately placed in cold PBS. If other tissues are to be harvested, they are also removed at this time and also placed in cold PBS. Collection of serum is facilitated by placing a borosilicate tube into the chest cavity in an angled position so that blood from the chest can drain into the tube. This tube is then placed on ice to allow clotting. Once all of the tissues have been harvested, they are trimmed and blotted dry. The tissues are weighed and transferred to Falcon 2059 tubes. The appropriate homogenization buffer is then added. We generally homogenize our tissues in 1ml buffer/OJg wet tissue weight. For CAT and luciferase assays we homogenize our samples in a buffer, which is prechilled on ice, composed of 25 mM glycylglycine (pH 7.8), 15 mM MgS04, 4 mM ethylene glycol bis(B-aminoethyl ether)-
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Fig. 1 Human growth hormone expression in adult rats followingdirect cardiac injection of plasmid DNA bearing the hGH gene (phGH) driven by the RSV promoter. Human growth hormone was
measured by radioimmunoassay in heart homogenate (left ventricle) or serum of the same animal
4 days after injection. N = 5.
N,N,N’,N’-tetraacetic acid (EGTA)(pH 7.8), and 1 mM dithiothreitol (DTT) (added at time of use). For hGH assays we homogenize our tissues in 0.25 M Tris, pH 7.4. Homogenization is performed using a Tissue Tek (Tekmar) homogenizer at the maximal setting for 20 sec. During homogenization, the sample is placed in a beaker of ice to keep it cool. The sample is then returned to an ice bucket while the remaining samples are homogenized. Generally, the homogenization is completed for one animal before the harvesting of tissues from the next animal begins. Once all samples have been homogenized, they are centrifuged at 5000s for 25 min at 4°C.Supernatants are then removed and measured. At this point samples can be placed at -70°C until they are ready to be assayed. We generally assay a fixed percentage of tissue from each sample. This is generally 5% of the lysate from constructs containing strong viral promoters; a larger percentage may need to be assayed when cellular promoters are used. In addition, when adenovirus is used it may be necessary to dilute the samples significantly. When assaying from AdCAT dilutions are made with 0.1 mg/ml BSA (bovine serum albumin).
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CAT and luciferase assays have been described in detail elsewhere (Kitsis et al., 1993). Briefly, when CAT assays are done alone the entire sample is heated to 65°C for 10 min. The lysates are cleared by microfuge centrifugation, and the assay is performed by the standard 14C-chloramphenicol,thin layer chromatography method (Kitsis et al., 1993). When CAT activity is being assayed along with luciferase or hGH, the sample is generally divided. Samples for both luciferase and hGH assays are kept on ice throughout the preparation. Luciferase assays are performed by diluting the sample corresponding to 5% of the lysate in homogenization buffer so that the final volume is 100 p1. Samples are usually done in duplicate; 360 pl of an ice-cold buffer containing 25 mM glycylglycine (pH 7.Q 15 mM MgS04, 4 mM EGTA (pH 7.8), 1 mM DTT, 15 mM potassium phosphate (mono- and dibasic, pH 7.8), 2 mM ATP (pH 7.0), and 0.27% (v/v) Triton X-100 is added to each sample. The final solution containing the substrate luciferin-02 mM luciferin (sodium salt), 25 mM glycylglycine (pH 7.8), 15 mM MgS04,4 mM EGTA (pH 7.8), 2 mM DTT-is added directly to the luminometer (Monolight 2010; Analytical Luminescence Laboratory, San Diego, California). Approximately 5 ml luciferin solution is enough for 30 samples. The luciferin solution should be kept in foil because of the light sensitivity of luciferin. The luminometer is adjusted to measure light production over 20 sec following the addition of the luciferin solution. A control tube containing only buffers should be assayed as well. Generally this background will be approximately 200-600 RLUs (relative light units). Growth hormone assays are done on either serum or homogenized tissue samples by radioimmunoassay. A kit containing standards and all materials is available from Nichols Institute Diagnostics (San Juan Capistrano, California). In our hands, this kit has produced very consistant results without any modification of their directions. Although we have not done quantitativej3-galactosidase assays, we have done j3-gal staining on tissue sections following direct injection. Many other groups have used recombinant adenoviruses with the &gal reporter gene in tissue sections as well (Jaffe et aZ., 1992, Le Gal La Salle et al., 1993). The p-gal staining is performed on frozen tissue sections. We freeze tissue in isopentane (Zmethylbutane) and store it at -70°C. The tissue is tembedded in OTC, and 10-pl sections are cut on a cryostat. The tissues are placed on poly (L-1ysine)coated slides and can be stored at -70°C. The sections are then fixed in 1.5% glutaraldehyde in PBS for 5 min at room temperature. The slides are then rinsed three times in PBS. The sections are then incubated in a solution containing 400 pg/ml X-gal, 5 mM potassium ferriferrocyanide, and 1 mM MgClz in PBS for approximately 6 hr.
IV.Experimental Design To minimize variability in expression from animal to animal, we have routinely used a second plasmid as an internal standard when comparing the strength of
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promoters. For example, when studying the effects of a cellular promoter driving the expression of luciferase, we have used a viral promoter driving CAT as a reference for injection quality. The promoter strength can then be reported as the ratio of luciferase activity to CAT activity. A twofold range of variability can still be seen following direct cardiac injection; therefore, we generally use five to eight animals per group. The use of two plasmids has brought to our attention a competition effect seen with strong viral promoters and large quantities of DNA. For example, when RSVCAT and RSVLUC are injected and are compared to an injection of RSVCAT and aLUC (alpha myosin heavy chain driving luciferase expression), higher levels of RSVCAT will be seen in the second group because it contains the weaker promoter (Buttrick et al., 1992). This phenomenon is presumably due to competition for transcription factors. We have found that this effect can be minimized when less plasmid is injected. For strong viral promoters, 0.52 p g of plasmid DNA should be sufficient to detect reporter gene expression. For each individual construct it may be necessary to test different doses in order to find the linear range. The minimum amount of plasmid that can be easily detected is best. To choose the best dose of adenovirus to inject, we quantitiated the level of CAT activity following intracardiac injection of various doses of AdCMVCATgD. For this virus, a roughly linear increase in CAT expression could be measured from 6 X lo6 pfu of virus through 2 X lo9 pfu, the highest dose tested. Adenovirus should be stored in a 50% glycerol solution. When highly concentrated virus was injected we saw an increase in mortality at the time of injection, presumably due to the concentration of glycerol in the injectate. We have routinely injected 6 X lo7 pfu of virus per animal. With our viral stocks, this is generally -1-10 pl virus stock, which can be diluted to 50 p1 with PBS. This dose gives us consistent results and limited mortality. Roughly fivefold variablity in expression can be seen in hearts 5 days following direct cardiac injection. Thoracic-cavityinjections generally give less consistent expression levels in a particular tissue, presumably because of the inherent imprecision of the route of administration. Using an ELISA assay for CAT protein (Boehringer-Mannheim), we have estimated that an average 132 pg of CAT protein can be expressed in the left ventricle 5 days following a single administration of adenovirus. In addition, approximately 20 ng/ml human growth hormone can be detected in serum following a single administration of AdhGH (secreted from all infected tissues), and 0.32 ng/ml of serum following hGH plamid injection. These quantities of protein are potentially consistent with a functional modification. Figure 2 shows the relative time course of expression from rats injected with either DNA or adenovirus. Following plasmid DNA injection, expression is maximal at approximately 7 days and remains consistent throughout the experiment. Animals injected with adenovirus show peak expression at approximately 5 days, but expression quickly drops, with no expression seen after 80 days. As
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Fig. 2 Time course of CAT expression following cardiac muscle injection of either pRSVCAT or AdCMVCAT. Adult rats were injected with either 100 pg RSVCAT plasmid DNA or 6 X lo7 pfu adenovirus expressing CAT. At various times after injection the left ventricles of each animal were homogenized and assayed for CAT expression. Relative CAT expressionis based on CAT expression in 5% of the tissue lysate multiplied by the dilution of the sample.
seen in Fig. 3, unlike in adults, expression of adenovirus in a neonate is stable with no significant change in expression through at least 7.5 months. The differences in duration of expression between adults and neonates, as well as between adenovirus and naked DNA, can be attributed to the generation of a severe immune response in adult animals following adenovirus administration. Five days after administration of adenovirus, high levels of neutralizing antibodiesare generated against the virus (Kass-Eisler et al., 1994).These neutralizing antibodies prohibit gene transfer from additional injections of adenovirus of the same serotype (Kass-Eisler ef al., 1994), making subsequent injections impossible. In addition to the antibody response, a severe infiltratinglymphocyte response can be seen in adenovirus-injected animals, suggesting T-cell involvement (Kass-Eider et d., 1993;Yang d d., 1994; Engelhardt et al., 1994). We and others are now trying to dissect the immune response to determine which factors
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Fig. 3 Time course of CAT expression in adult vs neonatal rats injected with 6 X lo7 pfu of AdCMVCAT. Adult rats were injected by intracardiac muscle injection. Neonatal rats were injected by thoracic cavity injection. Values are shown as relative CAT expression in the heart as in Fig. 2.
are involved in the transience of adenovirus-mediated gene expression in adults. Since these responses are not seen in neonatal animals given adenovirus, or in animals given plasmid DNA injections, these are currently better models for long-term gene transfer
V. Future Directions The advantageous features of naked DNA injection make it desirable to consider its optimization for research purposes and to consider its potential for therapeutics. This technique has already been hailed as a breakthrough for vaccines, and DNA vaccination has now been demonstrated to be effective in the laboratory setting (Ulmer et al., 1993; Fyran et aZ., 1993). The other system where DNA injection is likely to be effective therapeutically is in the expression of secreted gene products. As mentioned earlier, the use of DNA injection for studying cardiac gene regulation has already been shown to be effective (Kitsis et al., 1991; Buttrick et al., 1993), so for those purposes, no further modification
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of the technology needs to be considered. It is undoubtedly possible to increase the efficiency of gene transfer if the need arises. One potential means would be to increase the volume of the injectate. Another would be to test systematically the methods for preparing the DNA and the nature of the vehicle for delivery. A third potential means of optimization would be a mechanism for delivering multiple boluses of DNA over a wider geographic distribution. In contrast, the efficiency of adenovirus-mediated gene transfer is extremely high, and it is not considered to be necessary to improve it dramatically. However, major efforts are underway to develop more effective means of systemic delivery of virus. There are certain circumstances under which it might be desirable to have gene products expressed from multiple sites. Thus far, the most efficient means of achieving widespread gene transfer is to introduce virus into the apex of the left ventricle. This route of administration is even more efficient than when virus is introduced into the cardiac cavity (Kass-Eisler et al., 1994). The mechanism whereby injection of virus into the cardiac muscle is so efficient remains unknown, but is an observation that merits further investigation. The major hurdle that must be overcome with respect to adenovirus is the immune response to the virus. The immune response to the transgene must also be considered, although this would not be specific to adenoviruses as gene-transfer vehicles. The immune response to the virus appears to be both humoral and cellular and recognizes several structural proteins of the virus (Kass-Eisler et al., 1994;Engelhardt et al., 1994).Neutralizing antibodies to the virus develop rapidly after infection (within 5 days) and remain elevated for at least 96 days. In addition, there appears to be a potent cellular immune response, since inflammatory cells have been shown to be present in the area immediately surrounding infected cells (Yang et al., 1994; Engelhardt et al., 1994). Adenovirus DNA is lost as a function of time following infection, suggesting that infected cells are preferentially lost as a function of the immune system (Kass-Eisler et al., 1994). Two lines of experimentation are currently underway to solve this problem. The first relies upon the hypothesis that the immune response is activated primarily from leaky gene expression of late adenovirus genes, which are known to be potent immunogenes (Yang et al., 1994). This hypothesis has received support from the observation made by J. Wilson’s group that a recombinant adenovirus with a temperature-sensitivemutation in the E2b gene (in addition to an Ela deletion), which has a regulatory role in late gene expression, shows a longer duration of gene expression than its Ela-deleted counterpart (Engelhardt et al., 1994). However, it remains to be formally proven that late gene expression is responsible for the transient expression of adenovirus-encodedgenes. Further manipulations of the viral genome are ongoing to eliminate additional viral genes. This will require the creation of cell lines that can supply those functions in trans for the production of viruses. The second line of experimentation that addresses these issues is to define which aspects of the immune system are responsible for the transient expression of genes. For these purposes, our laboratory is determining the duration of
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expression following injection of the AdCAT virus in mice that have been genetically manipulated to “knock out” various aspects of the immune systems. Preliminary results suggest that the immune system involvement may be different in different tissues (Kass-Eisler, Bloom, and Leinwand, unpublished observation) and that both humoral and cellular immunity are involved in the temporal decline in gene expression. Once the determinants of the immune system are defined, it may be possible to devise local and temporally restricted immunosuppressive agents that allow long-term gene expression to occur. Additional strategies for long-term, efficient gene transfer are also being developed using viral fragments and chemical reagents. To take advantage of the ability of adenovirus to disrupt the endosome, a number of groups have successfullyused adenovirus either chemically coupled or mixed with plasmid DNA (Curiel et af., 1991; Cotten et af., 1992; Yoshimura et af., 1993) in cultured cells. Both fetal and adult cardiac myocytes in culture can be transfected in this manner (Rivera and Leinwand, unpublished observation). These experiments now need to be performed in the intact animal to test whether efficient gene transfer can be accomplished by this method. The major advantage of this technology is that the adenoviruses used in these types of experiments can be psoralen or UVinactivated, therefore reducing the possibility of viral gene expression, and potentially reducing the immune response (Cotten et al., 1992). If the immune response to adenovirus cannot be overcome, then nonviral substitutes must be developed for long-term efficient gene transfer. One promising report (Zhu et al., 1993) has shown that when mixed with cationic liposomes, DNA transfer into adult mice by intravenous injection is relatively efficient and expression can last for at least 9 weeks. The field of gene transfer is one that is receiving a great deal of attention, and substantial progress has been made in a relatively short time. Many of the areas in need of improvement or expansion have now been identified, and it seems clear that in the near future, safe and effective gene transfer will be in common use. References Ascadi, G., Jiao, S. S., Jani, A,, Duke, D., Williams, P., Chong, W., and Wolff, J. A. (1991).Direct gene transfer and expression into rat heart in vivo. New Biol. 3,71-81. Bett, A. J., Haddara, W., Prevec, L., and Graham, F. L. (1994). An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3. Proc. Nafl. Acad. Sci. USA 91,8802-8806. Buttrick, P. M., Kass, A., Kitsis, R. N., Kaplan, M. L., and Leinwand, L. A. (1992). Behavior of genes directly injected into rat heart in vivo. Circ.Res. 70, 193-198. Buttrick, P. M.,Kaplan, M. L., Kitsis, R. N., and Leinwand L. A. (1993). Distinct behavior of cardiac myosin heavy chain gene constructs in vivo-discordance with in vitro results. Circ. Res.
72,1211-1217.
Cotten, M., Wagner, E., Zatloukal, K.,Phillips, S., Curiel, D. T., and Birnstiel, M. L. (1992).Highefficiency receptor-mediated delivery of small and large (48 kilobase) gene constructs using the endosome-disruption activity of defective or chemically inactivated adenovirus particles. Proc. Natl. Acad. Sci. USA 89,6094-6098.
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Curiel,D. T., Agarwal,S., Wagner,E. and Cotten, M. (1991). A d e n o h enhancement of transferrinpolylysine-mediated gene delivery. Proc. NatL Acad. Sci. USA 88,8850-8854. Engelhardt, J. F., Ye, X.,Doranz, B., and Wilson, J. M. (1994). Ablation of E2A in recombinant adenoviruses improves transgene persistence and decreases inflammatory response in mouse liver. Proc. Natl. Acad. Sci. USA 91,61%-6200. Fynan, E. F., Webster, R. G., Fuller, D. H., Haynes, J. R., Santoro, J. C., and Robinson, H. L. (1993). DNA vaccines: Protective immunizations by parenteral, mucosal, and gene-gun innoculations. Proc. Natl. Acad. Sci USA 90,11478-11482. Hansen, E., Fernandes, K., Goldspink, G., Butterworth, P., Umeda, P. K., and Chang, K. C. (1991). Strong expression of foreign genes following direct injection into fish muscle. FEBS Letfers 290, 73-76. Jaffe, H. A., Danel, C., Longenecker, G., Metzger, M., Setouchi, Y., Rosenfeld, M. A., Gant, T. W., Thorgeirsson, S. S., Stratford-Pemcaudet, L. S., Pemcaudet, M., Pavirani, A., Lecocq, J.-P., and Crystal, R. G. (1992). Adenovims-mediated in vivo gene transfer and expression in normal rat liver. Nature Genetics 1,372-378. Kass-Eisler,A,, Falck-Pedersen,E., Alvira, M., Rivera, J., Buttrick, P. M., Wittenberg, B. A., Cipriani, L., and Leinwand, L. A. (1993). Quantitative determination of adenovirus-mediatedgene delivery to rat cardiac myocytes in vitro and in vivo. Proc. Natl. Acad. Sci. USA 90,11498-11502. Kass-Eisler, A., Falck-Pedersen, E., Elfenbein, D. H., Alvira, M., Buttrick, P. M., and Leinwand, L. A. (1994). Distribution, duration, and immune response to adenovirus-mediated gene transfer. Gene Therapy 1,395-402. Kitsis, R. N., Buttrick, P. M., McNally, E. M., Kaplan, M. L., and Leinwand L. A. (1991). Hormonal modulation of a gene injected into rat heart in vivo. Proc. Natl. Acad. Sci. USA 88,4138-4142. Kitsis, R. N., Buttrick, P. M., Kass, A. A., Kaplan, M. L., and Leinwand L. A. (1993). Gene transfer into adult rat heart in vivo. Methods in Molecular Genetics 1,374-392. Le Gal La Salle, G., Robert, J. J., Berrard, S., Ridoux, V.,Stratford-Perricaudet, L. D., Perricaudet, M., and Mallet, J. (1993). An adenovirus vector for gene transfer into neurons and glia in the brain. Science 259, 988-990. Lin, H., Parmacek, M.S., Morle, G., Bollling, S., and Leiden, J. M. (1990). Expression of recombinant genes in myocardium in vivo after direct injection of DNA. Circulation 82,2217-2221. Mittal, S . K., McDermott, M. R., Johnson, D. C., Prevec, L., and Graham, F. L. (1993). Monitoring foreign gene expression by a human adenovirus-based vector usning the firefly luciferase gene as a reporter. Virus Research 28,67-90. Quantin, B., Pemcaudet, L. D., Tajbakhsh, S., and Mandel, J-L. (1991). Adenovirus as an expression vector in muscle cells in vivo. Proc. Natl. Acad. Sci. USA 89,2581-2584. Rosenfeld, M. A., Siegfried, W., Yoshiura, K., Yoneyama, K., Fukayama, M., Stier, L. E., Paakko, P. K., Gilardi, P., Stratford-Pemcaudet, L. D., Pemcaudet, M., Jallat, S., Pavirani, A., Lecocq, J.-P., and Crystal, R. G. (1991). Adenovirus-mediated transfer of a recombinant al-antitrypsin gene to the lung epithelium in vivo. Science 252,431-434. Selden, R. F., Howie, K. B., Rowe, M. E., Goodman, H. M., and Moore, D. D. (1986). Human growth hormone as a reporter gene in regulation studies employing transient gene expression. Mol. Cell Biol. 6,3173-3179. Stratford-Pemcaudet, L. D., Makeh, I., Pemcaudet, M., and Briand, P. (1992). Widespread longterm gene transfer to mouse skeletal muscles and heart. J. Clin. Invest. 90, 626-630. Ulmer, J. B., Donelly, J. J., Parker, S. E., Rhodes, G. H., Felgner, P. L., Dwarki, V.J., Gromkowski, S. H., Deck, R. R., De Witt, C. M., Friedman, A., Hawe, L. A., Leander, K. R., Martinez, D., Perry, H. C., Shirer, J. W., Montgomery, D. C., and Liu, M. A. (1993). Heterologous protection against influenza by injection of DNA encoding a viral protein. Science 259,1745-1749. von Harsdorf, R., Schott, R. J., Shen, Y. T., Vatner, S. F., Mahdavi, V.,and Nadal-Ginard, B. (1993). Gene injection into canine myocardium as a useful model for studying gene expression in the heart of large mammals. Circ. Res. 72,688-695. Wolff, J. A., Malone, R. W., Williams, P., Chong, W., Ascadi, G., Jani, A., and Felgner, P. L. (1990). Direct gene transfer into mouse muscle in vivo. Science 247, 1465-1468.
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Wolff, J. A., Williams, P., Ascadi, G., Jiao, S.,Jani, A., and Chong, W. (1991). Conditions affecting direct gene transfer into rodent muscles in vivo. Biorechniques 11,474-485. Wolff, J. A., Ludtke, J. J., Acsadi, G., Williams, P., and Jani, A. (1992). Long-term persistence of plasmid DNA and foreign gene expression in mouse muscle. Human Molecular Genetics 1,363-369. Yang, Y.,Nunes, F. A., Berenni, K., Furth, E. E., Gonnol, E., and Wilson,J. M. (1994). Cellular immunity to viral antigens limits El-deleted adenoviruses for gene therapy. Proc. Natl. Acad. Sci. USA 91,4407-4411. Yoshimura, K., Rosenfeld, M. A., Seth, P., and Crystal, R. G. (1993). Adenovirus-mediated augmentation of cell transfection with unmodified plamid vectors. J. B i d Chem. 268,2300-2303. Zhu, N., Liggitt, D., Liu, Y.,and Debs, R. (1993). Systemic gene expression after intravenous DNA delivery into adult mice. Science 261,209-211.
DNA- and Adenovirus-Mediated Gene Transfer into Cardiac Muscle Alyson Kass-Eisler* and Leslie A. Leinwandt 'Cold Spring Harbor Laboratory Cold Spring Harbor, New York 11724 +Department of Molecular, Cellular, and Developmental Biology University of Colorado at Boulder Boulder. Colorado 80309
I. Introduction 11. Methods for Cardiac Injection 111. Homogenization of Tissue and Assays IV. Experimental Design V. Future Directions References
I. Introduction In this chapter we will compare two methods of gene transfer into the rodent heart: direct plasmid DNA injection and injection of recombinant adenovirus. The goals of cardiac gene transfer that will be considered here are defining the elements regulating cardiac gene expression and phenotypic modification. Most studies aimed at understanding cardiac gene regulation have used transient transfection of DNA constructs into fetal or neonatal cardiocyte cultures. No transfections of adult cardiocytes have been reported. However, these studies are limited because of the developmental stage difference between the cultured cells and the adult and because of the inability to reproduce complex physiological and pathological processes in a tissue-culture environment. One solution to this problem is the creation of transgenic mice bearing reporter genes whose expression is driven by various promoters and putative regulatory regions. However, the creation of transgenic animals for this purpose is both time-consuming and expenMETHODS IN CELL BIOLOGY. VOL. 52 Copyright Q 1998 by Academic Press. AU rights of reproduction in any form resewed. lN91-67YX/98 125.00
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sive. Direct gene injection using naked plasmid DNA was first described in skeletal muscle by J. Wolff et al. in 1990. Several laboratories have extended the use of this methodology to include heart, which appears to be much more efficient than skeletal muscle in taking up and/or expressing naked DNA (Lin et al., 1990; Ascadi et al., 1991; Buttrick et al., 1992). The predominant use of direct injection into the heart has been to analyze promoter elements in vivo using reporter genes driven by the promoter element of interest (Kitsis et al., 1991; Buttrick et al., 1993). There are several advantageous features of naked DNA-mediated gene transfer. Primary among these are the stable expression resulting from gene transfer (Wolff et al., 1992) and the relative simplicity of the approach, which avoids infectious agents. Reports of gene expression beyond 1 year exist (Wolff et al., 1992). In addition, the DNA remains episomal, avoiding potential risks associated with the integration of DNA into the host genome (Wolff et al., 1992). The major drawback to this approach is its relative inefficiency.The maximum activity that can be obtained from gene expression appears to be restricted to an area immediately surrounding the injection site (Buttrick et al., 1992). Furthermore, the number of cells that take up and express the DNA is relatively small, corresponding to -0.02% of the myocytes in the heart (Kitsis et al., 1993). These drawbacks do not affect the utility of this method for mapping the regulatory elements of genes. Substantial reporter gene activity is obtained even after the introduction of relatively small amounts of DNA. In fact, when contemplating gene regulation studies, it is important to use relatively small quantities of DNA if the promoter is very active. For example, a strong promoter such as the Rous sarcoma virus LTR is out of the linear range of dose responsiveness above -2 pg in a rat heart. The assumption is that there are limiting transcription factors and that the cell rapidly becomes saturated with DNA sequences that bind these factors. Therefore, a DNA dose-response curve should be generated for each promoter under investigation. The second general use of in vivo gene transfer is to examine the consequences of expression of a given gene on the structure or function of the heart. This could involve expression of a mutant protein or ectopic expression of an isoform of a protein normally found in another setting. For these purposes, unless the gene encodes a secreted protein, DNA injection would not be efficient enough for most forms of phenotypic modification. We have therefore turned to the use of recombinant adenoviruses. Adenoviruses are double-stranded linear DNA viruses that have been extensively characterized in clinical and laboratory settings. Adenoviruses efficiently infect a large number of replicating and nonreplicating cell types. Their ability to infect nonreplicating cells makes them ideal for use in the heart, since adult cardiac myocytes are terminally differentiated. The recombinant adenoviruses in current use by most laboratories are replication deficient because they are deleted in a major regulatory region, termed Ela. Vectors currently exist that can accommodate up to 8 kb of a recombinant sequence, which makes them potentially useful for the majority of cDNA se-
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quences (Bett et af., 1994). These viruses can also be produced as extremely high-titer stocks (1011-1012pfdml). Finally, adenoviruseshave an excellent safety profile. The genome rarely integrates into the host genome and has never been associated with human malignancy. Several groups have demonstrated very efficient infection of the cardiovascular system by recombinant adenoviruses (Kass-Eisler et af., 1993; Stratford Perricaudet et al., 1992). Our group has focused on direct intracardiacmuscle injection of viruses. We first determined that both fetal and adult cardiac myocytes can be efficiently infected in culture (Kass-Eisler et af., 1993), achieving virtually 100% infection with a multiplicity of infection (MOI) of 2-3 pfdcell. We then examined the amount and distribution of gene expression resulting from injection of various viral doses into the left ventricular wall of the rat heart. Between 6 X lo6 and 2 X lo9 pfu there is a linear response between viral dose and expression. We have also compared the efficiency of gene transfer of a plasmid and an adenovirus bearing identical transgenes. For these experiments, we have used an adenovirus of the serotype 5 that bears the CAT reporter gene driven by the cytomegaloviruspromoter. The CAT gene was chosen because of its ability to be quantitated as well as its ability to be detected immunohistochemically.At maximal dose responsiveness for virus and plasmid DNA, the amount of CAT activity was approximately lOOOX higher in hearts injected with virus, 5 days following injection (Kass-Eisler et af., 1993), compared with its level in hearts injected with plasmid DNA. We had previously demonstrated in hearts injected with DNA that 98% of the activity of reporter genes was restricted to a small area around the injection site and that the transfection efficiency was low, corresponding to only -2000 cells in the entire heart (Kitsis et al., 1993). We compared both the distribution and the proportion of CAT-positive cells resulting from a single virus injection with results obtained from DNA injection. The quantitative distribution of expression was no different between adenovirus and plasmid DNA injection, in that the majority of expression was restricted to the injection site. However, because the amount of gene expression was so much higher in the virus injections, there was significant gene expression throughout the entire heart. In order to determine the proportion of cells in the heart which were infected with the virus, sections of the heart were stained with an anti-CAT antibody. In the regions of the heart surrounding the injection site, virtually 100% of cells were expressing CAT. In contrast to naked DNA injection, which transfects only cardiac myocytes, all cell types in the heart were able to be infected by the virus. The proportion of CAT-positive cells decreased as a function of distance from the injection site, reaching -5-10% of the cells at the base of the heart, which was the most distal location relative to the injection site. An additional feature of adenovirus infections in the adult heart that must be taken into consideration is the transient expression of genes. A universal observation with adenovirus is that independently of the route of the virus or the transgene it expresses, expression is transient, dropping sharply within -2 weeks, reaching undetectable levels
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by 80 days (Kass-Eisler et al., 1994; Mittal et al., 1993; Quantin et al., 1991). It now seems clear that the long-term expression of genes is limited by the immune response of the host to the virus and to the transgene. This immune response appears to be both humoral and cellular (Kass-Eisler et al., 1994; Yang et al., 1994). Extremely high levels of neutralizing antibodies develop within 5 days following virus administration (Kass-Eisler et al., 1994). Antibodies directed against the transgene also develop, but with a temporal delay compared with those directed against the virus (Kass-Eisler et aL, 1994).Traditionalimmunosuppression by agents such as cyclosporine is ineffective in prolonging gene expression (Kass-Eisler et al., 1994; Engelhardt et al., 1994). Experiments are underway to examine the time course of gene expression in transgenic animals whose immune systems have been modified in a variety of ways. The response of the immune system could be potentially circumvented by introducing virus into the immunologicallyincompetent neonatal animal. Neonatal rodents are immunologically incompetent and therefore do not recognize viral or transgene proteins as foreign. Expression by adenovirus-mediated genes in a neonate appears to be indefinite, reaching a time course of at least 7-8 months (Kass-Eisler et al., 1994; Rosenfeld et al., 1991). Experiments are currently in progress to determine whether neonates can be tolerized to the virus, allowing multiple administration of various recombinant viruses. In the case of injection into neonates, the technique will be described later. Because of the size of the animals, direct cardiac injection is not feasible. Therefore, animals are injected percutaneously into the thoracic cavity. This mode of introducing virus results in effective infection of multiple organs, including heart, diaphragm, lung, and liver, for an indefinite period of time. The amount of CAT activityper heart is approximately 1OOOX lower than that seen from direct cardiac injection. The lower levels of expression in the heart are most likely due to the distribution of virus by this route of administration. Thoracic-cavity injection most likely results in the majority of adenovirus being introduced into the lung. In support of this, 100-fold greater expression levels can be measured in the lungs of neonatally injected animals than in the lungs of directly cardiac-injected adults (Kass-Eisler et al., 1994).
II. Methods for Cardiac Injection Our injection protocol involves a simple surgical procedure to exteriorize the heart followed by injection of the heart muscle with a hypodermic needle and is usually performed by two people. Although other injection protocols have been developed for various species (Hansen et aZ., 1991; von Harsdorf et aL, 1993), the following protocol is for adult rats. This procedure can be used for delivery of naked DNA as well as recombinant adenoviruses. Animals are anesthetized with an intraperitoneal injection of 4% chloral hydrate (made fresh with water) at a dosage 0.75 d l 0 0 g body weight. This
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anesthesia, which takes effect within -10 min, lasts for approximately 30 min of deep sleep followed by approximately 1-2 hr of partial anesthesia. When animals are properly anesthetized there should be a loss of corneal reflexes and a lack of withdrawal to painful stimulation. Once anesthetized the animal is laid in a supine position. A vertical incision is made slightly to the left of midline with either a scalpel or sharp scissors. This can be accomplished through a natural slit in the muscles that runs diagonally across the animal’s left chest. The chest muscles are then blunt-dissected to the level of the ribs. The apical impulse of the heart can then be visualized (avoid the pulsation coming from the nearby great vessels). The rib just below the apical impulse is then cut with a scissors 5 mm to the left of the sternum. An incision too close to the sternum would risk damaging the internal mammary artery. The scissorsshould be held perpendicular and inserted as shallowly as possible to avoid damage to the heart or lungs. A Crile forceps is then inserted into the chest cavity through the rib incision perpendicular to the chest wall. The incision is then gently widened with the opening of the forceps. This opening of the chest cavity will cause the lungs to deflate, preventing respiration; therefore, it is important to continue as quickly as possible. The heart can usually be seen in the chest cavity at this point. If not, gentle pressure the right side of the chest can help move the heart into view. The heart is then gently exteriorized by grasping its apex with the Crile forceps. It is very important not to grip the heart by the atria or the great vessels, or to grasp the lungs. The heart can then be gently held by the operator’s fingers while the second operator performs the injection. A 100-pl Hamilton syringe with a 27-gauge needle containing the injectate is then inserted into the wall of the left ventricle. We have generally used an injectate volume of 50 p1 PBS (137 mM NaCl; 2.7 mM KCl; 10 mM Na2HP04; 2 mM KH2P04,pH 7.4)’ but this volume can be varied from 10 to 100 pl (Wolff et al., 1991) if necessary for a rat heart. The heart is then gently placed back into the chest cavity. The chest of the animal is then gently squeezed with fingers to expel any air from the cavity. The lungs are inflated using a Harvard small-animal respirator with a nose-cone adapter that fits over the snout of the rat. The nose cone is easily constructed by cutting of the bottom 4 cm of a 30-ml syringe and attaching a piece of glove latex over the wide end. The cone is attached to the respirator through tubing placed on the Luer-lock end of the syringe. The need for intubating the trachea is eliminated with the use of the nose cone, which provides positive-pressure ventilation of the animal. Two to four respirations with the respirator should be enough to induce spontaneous respiration; additional use should be kept to a minimum. At this point the muscle layers of the chest are closed using 3-0 silk sutures. Complete evacuation of pneumothorax should be accomplished before final suturing of the chest by gentle squeezing of the chest. The skin can then be stapled closed. Approximately 10%of all animals will succumb during this surgical
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procedure. The two main causes of death are improper anesthetic dosage and the trapping of air in the chest. Unlike rats, mice do not survive this procedure as well without modification. We have recently injected mice with adenovirus directly into the thoracic cavity. To do this, mice are held gently between the thumb and forefinger, and a 26gauge needle containing virus in a volume of 30 pl is introduced into the chest near the site of the heart. Care must be taken to insert and remove the needle slowly and gently. Complete immobilization of the animal is essential. Approximately 10% mortality (usually immediately after injection) can be seen with this procedure. Thoracic-cavity injection is also quite effective in the infection of neonatal animals. Neonatal animals are quite transparent, and if they are held over a light source, the ability to inject near the heart is greatly enhanced. Mortality rates from the injection in neonates are only about 2%.
III. Homogenization of Tissue and Assays For CAT (chloramphenicol acetyl transferase) and luciferase assays, tissues are homogenized. We have also used /3-galactosidase as a reporter gene, in which case whole tissues are harvested for staining. In addition, we have used human growth hormone (Selden et aZ., 1986) as a reporter gene in rats. Although high levels of expression of hGH can be measured in heart following administration of either phGH or AdhGH by direct cardiac injection,there is very little secretion of the hormone from the heart (see Fig. l),although detectable amounts in serum allow for multiple measurements on the same animal over time. In addition, hGH activity can be measured in homogenized tissue as discussed later. Before sacrifice, animals are given a large dose of 4% chloral hydrate (-1.5 ml/lOO g body weight). This is potentially a lethal dose, so care should be taken not to let the animals expire before the removal of tissues. The heart is removed by an abdominal incision through the midline. The ribs on both sides of the xiphoid process of the sternum are cut, revealing the heart in the chest cavity. The heart is then held with forceps by the apex while being cut at the base of the heart with a pair of scissors. The heart is immediately placed in cold PBS. If other tissues are to be harvested, they are also removed at this time and also placed in cold PBS. Collection of serum is facilitated by placing a borosilicate tube into the chest cavity in an angled position so that blood from the chest can drain into the tube. This tube is then placed on ice to allow clotting. Once all of the tissues have been harvested, they are trimmed and blotted dry. The tissues are weighed and transferred to Falcon 2059 tubes. The appropriate homogenization buffer is then added. We generally homogenize our tissues in 1ml buffer/OJg wet tissue weight. For CAT and luciferase assays we homogenize our samples in a buffer, which is prechilled on ice, composed of 25 mM glycylglycine (pH 7.8), 15 mM MgS04, 4 mM ethylene glycol bis(B-aminoethyl ether)-
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Fig. 1 Human growth hormone expression in adult rats followingdirect cardiac injection of plasmid DNA bearing the hGH gene (phGH) driven by the RSV promoter. Human growth hormone was
measured by radioimmunoassay in heart homogenate (left ventricle) or serum of the same animal
4 days after injection. N = 5.
N,N,N’,N’-tetraacetic acid (EGTA)(pH 7.8), and 1 mM dithiothreitol (DTT) (added at time of use). For hGH assays we homogenize our tissues in 0.25 M Tris, pH 7.4. Homogenization is performed using a Tissue Tek (Tekmar) homogenizer at the maximal setting for 20 sec. During homogenization, the sample is placed in a beaker of ice to keep it cool. The sample is then returned to an ice bucket while the remaining samples are homogenized. Generally, the homogenization is completed for one animal before the harvesting of tissues from the next animal begins. Once all samples have been homogenized, they are centrifuged at 5000s for 25 min at 4°C.Supernatants are then removed and measured. At this point samples can be placed at -70°C until they are ready to be assayed. We generally assay a fixed percentage of tissue from each sample. This is generally 5% of the lysate from constructs containing strong viral promoters; a larger percentage may need to be assayed when cellular promoters are used. In addition, when adenovirus is used it may be necessary to dilute the samples significantly. When assaying from AdCAT dilutions are made with 0.1 mg/ml BSA (bovine serum albumin).
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CAT and luciferase assays have been described in detail elsewhere (Kitsis et al., 1993). Briefly, when CAT assays are done alone the entire sample is heated to 65°C for 10 min. The lysates are cleared by microfuge centrifugation, and the assay is performed by the standard 14C-chloramphenicol,thin layer chromatography method (Kitsis et al., 1993). When CAT activity is being assayed along with luciferase or hGH, the sample is generally divided. Samples for both luciferase and hGH assays are kept on ice throughout the preparation. Luciferase assays are performed by diluting the sample corresponding to 5% of the lysate in homogenization buffer so that the final volume is 100 p1. Samples are usually done in duplicate; 360 pl of an ice-cold buffer containing 25 mM glycylglycine (pH 7.Q 15 mM MgS04, 4 mM EGTA (pH 7.8), 1 mM DTT, 15 mM potassium phosphate (mono- and dibasic, pH 7.8), 2 mM ATP (pH 7.0), and 0.27% (v/v) Triton X-100 is added to each sample. The final solution containing the substrate luciferin-02 mM luciferin (sodium salt), 25 mM glycylglycine (pH 7.8), 15 mM MgS04,4 mM EGTA (pH 7.8), 2 mM DTT-is added directly to the luminometer (Monolight 2010; Analytical Luminescence Laboratory, San Diego, California). Approximately 5 ml luciferin solution is enough for 30 samples. The luciferin solution should be kept in foil because of the light sensitivity of luciferin. The luminometer is adjusted to measure light production over 20 sec following the addition of the luciferin solution. A control tube containing only buffers should be assayed as well. Generally this background will be approximately 200-600 RLUs (relative light units). Growth hormone assays are done on either serum or homogenized tissue samples by radioimmunoassay. A kit containing standards and all materials is available from Nichols Institute Diagnostics (San Juan Capistrano, California). In our hands, this kit has produced very consistant results without any modification of their directions. Although we have not done quantitativej3-galactosidase assays, we have done j3-gal staining on tissue sections following direct injection. Many other groups have used recombinant adenoviruses with the &gal reporter gene in tissue sections as well (Jaffe et aZ., 1992, Le Gal La Salle et al., 1993). The p-gal staining is performed on frozen tissue sections. We freeze tissue in isopentane (Zmethylbutane) and store it at -70°C. The tissue is tembedded in OTC, and 10-pl sections are cut on a cryostat. The tissues are placed on poly (L-1ysine)coated slides and can be stored at -70°C. The sections are then fixed in 1.5% glutaraldehyde in PBS for 5 min at room temperature. The slides are then rinsed three times in PBS. The sections are then incubated in a solution containing 400 pg/ml X-gal, 5 mM potassium ferriferrocyanide, and 1 mM MgClz in PBS for approximately 6 hr.
IV.Experimental Design To minimize variability in expression from animal to animal, we have routinely used a second plasmid as an internal standard when comparing the strength of
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promoters. For example, when studying the effects of a cellular promoter driving the expression of luciferase, we have used a viral promoter driving CAT as a reference for injection quality. The promoter strength can then be reported as the ratio of luciferase activity to CAT activity. A twofold range of variability can still be seen following direct cardiac injection; therefore, we generally use five to eight animals per group. The use of two plasmids has brought to our attention a competition effect seen with strong viral promoters and large quantities of DNA. For example, when RSVCAT and RSVLUC are injected and are compared to an injection of RSVCAT and aLUC (alpha myosin heavy chain driving luciferase expression), higher levels of RSVCAT will be seen in the second group because it contains the weaker promoter (Buttrick et al., 1992). This phenomenon is presumably due to competition for transcription factors. We have found that this effect can be minimized when less plasmid is injected. For strong viral promoters, 0.52 p g of plasmid DNA should be sufficient to detect reporter gene expression. For each individual construct it may be necessary to test different doses in order to find the linear range. The minimum amount of plasmid that can be easily detected is best. To choose the best dose of adenovirus to inject, we quantitiated the level of CAT activity following intracardiac injection of various doses of AdCMVCATgD. For this virus, a roughly linear increase in CAT expression could be measured from 6 X lo6 pfu of virus through 2 X lo9 pfu, the highest dose tested. Adenovirus should be stored in a 50% glycerol solution. When highly concentrated virus was injected we saw an increase in mortality at the time of injection, presumably due to the concentration of glycerol in the injectate. We have routinely injected 6 X lo7 pfu of virus per animal. With our viral stocks, this is generally -1-10 pl virus stock, which can be diluted to 50 p1 with PBS. This dose gives us consistent results and limited mortality. Roughly fivefold variablity in expression can be seen in hearts 5 days following direct cardiac injection. Thoracic-cavityinjections generally give less consistent expression levels in a particular tissue, presumably because of the inherent imprecision of the route of administration. Using an ELISA assay for CAT protein (Boehringer-Mannheim), we have estimated that an average 132 pg of CAT protein can be expressed in the left ventricle 5 days following a single administration of adenovirus. In addition, approximately 20 ng/ml human growth hormone can be detected in serum following a single administration of AdhGH (secreted from all infected tissues), and 0.32 ng/ml of serum following hGH plamid injection. These quantities of protein are potentially consistent with a functional modification. Figure 2 shows the relative time course of expression from rats injected with either DNA or adenovirus. Following plasmid DNA injection, expression is maximal at approximately 7 days and remains consistent throughout the experiment. Animals injected with adenovirus show peak expression at approximately 5 days, but expression quickly drops, with no expression seen after 80 days. As
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Fig. 2 Time course of CAT expression following cardiac muscle injection of either pRSVCAT or AdCMVCAT. Adult rats were injected with either 100 pg RSVCAT plasmid DNA or 6 X lo7 pfu adenovirus expressing CAT. At various times after injection the left ventricles of each animal were homogenized and assayed for CAT expression. Relative CAT expressionis based on CAT expression in 5% of the tissue lysate multiplied by the dilution of the sample.
seen in Fig. 3, unlike in adults, expression of adenovirus in a neonate is stable with no significant change in expression through at least 7.5 months. The differences in duration of expression between adults and neonates, as well as between adenovirus and naked DNA, can be attributed to the generation of a severe immune response in adult animals following adenovirus administration. Five days after administration of adenovirus, high levels of neutralizing antibodiesare generated against the virus (Kass-Eisler et al., 1994).These neutralizing antibodies prohibit gene transfer from additional injections of adenovirus of the same serotype (Kass-Eisler ef al., 1994), making subsequent injections impossible. In addition to the antibody response, a severe infiltratinglymphocyte response can be seen in adenovirus-injected animals, suggesting T-cell involvement (Kass-Eider et d., 1993;Yang d d., 1994; Engelhardt et al., 1994). We and others are now trying to dissect the immune response to determine which factors
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Fig. 3 Time course of CAT expression in adult vs neonatal rats injected with 6 X lo7 pfu of AdCMVCAT. Adult rats were injected by intracardiac muscle injection. Neonatal rats were injected by thoracic cavity injection. Values are shown as relative CAT expression in the heart as in Fig. 2.
are involved in the transience of adenovirus-mediated gene expression in adults. Since these responses are not seen in neonatal animals given adenovirus, or in animals given plasmid DNA injections, these are currently better models for long-term gene transfer
V. Future Directions The advantageous features of naked DNA injection make it desirable to consider its optimization for research purposes and to consider its potential for therapeutics. This technique has already been hailed as a breakthrough for vaccines, and DNA vaccination has now been demonstrated to be effective in the laboratory setting (Ulmer et al., 1993; Fyran et aZ., 1993). The other system where DNA injection is likely to be effective therapeutically is in the expression of secreted gene products. As mentioned earlier, the use of DNA injection for studying cardiac gene regulation has already been shown to be effective (Kitsis et al., 1991; Buttrick et al., 1993), so for those purposes, no further modification
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of the technology needs to be considered. It is undoubtedly possible to increase the efficiency of gene transfer if the need arises. One potential means would be to increase the volume of the injectate. Another would be to test systematically the methods for preparing the DNA and the nature of the vehicle for delivery. A third potential means of optimization would be a mechanism for delivering multiple boluses of DNA over a wider geographic distribution. In contrast, the efficiency of adenovirus-mediated gene transfer is extremely high, and it is not considered to be necessary to improve it dramatically. However, major efforts are underway to develop more effective means of systemic delivery of virus. There are certain circumstances under which it might be desirable to have gene products expressed from multiple sites. Thus far, the most efficient means of achieving widespread gene transfer is to introduce virus into the apex of the left ventricle. This route of administration is even more efficient than when virus is introduced into the cardiac cavity (Kass-Eisler et al., 1994). The mechanism whereby injection of virus into the cardiac muscle is so efficient remains unknown, but is an observation that merits further investigation. The major hurdle that must be overcome with respect to adenovirus is the immune response to the virus. The immune response to the transgene must also be considered, although this would not be specific to adenoviruses as gene-transfer vehicles. The immune response to the virus appears to be both humoral and cellular and recognizes several structural proteins of the virus (Kass-Eisler et al., 1994;Engelhardt et al., 1994).Neutralizing antibodies to the virus develop rapidly after infection (within 5 days) and remain elevated for at least 96 days. In addition, there appears to be a potent cellular immune response, since inflammatory cells have been shown to be present in the area immediately surrounding infected cells (Yang et al., 1994; Engelhardt et al., 1994). Adenovirus DNA is lost as a function of time following infection, suggesting that infected cells are preferentially lost as a function of the immune system (Kass-Eisler et al., 1994). Two lines of experimentation are currently underway to solve this problem. The first relies upon the hypothesis that the immune response is activated primarily from leaky gene expression of late adenovirus genes, which are known to be potent immunogenes (Yang et al., 1994). This hypothesis has received support from the observation made by J. Wilson’s group that a recombinant adenovirus with a temperature-sensitivemutation in the E2b gene (in addition to an Ela deletion), which has a regulatory role in late gene expression, shows a longer duration of gene expression than its Ela-deleted counterpart (Engelhardt et al., 1994). However, it remains to be formally proven that late gene expression is responsible for the transient expression of adenovirus-encodedgenes. Further manipulations of the viral genome are ongoing to eliminate additional viral genes. This will require the creation of cell lines that can supply those functions in trans for the production of viruses. The second line of experimentation that addresses these issues is to define which aspects of the immune system are responsible for the transient expression of genes. For these purposes, our laboratory is determining the duration of
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expression following injection of the AdCAT virus in mice that have been genetically manipulated to “knock out” various aspects of the immune systems. Preliminary results suggest that the immune system involvement may be different in different tissues (Kass-Eisler, Bloom, and Leinwand, unpublished observation) and that both humoral and cellular immunity are involved in the temporal decline in gene expression. Once the determinants of the immune system are defined, it may be possible to devise local and temporally restricted immunosuppressive agents that allow long-term gene expression to occur. Additional strategies for long-term, efficient gene transfer are also being developed using viral fragments and chemical reagents. To take advantage of the ability of adenovirus to disrupt the endosome, a number of groups have successfullyused adenovirus either chemically coupled or mixed with plasmid DNA (Curiel et af., 1991; Cotten et af., 1992; Yoshimura et af., 1993) in cultured cells. Both fetal and adult cardiac myocytes in culture can be transfected in this manner (Rivera and Leinwand, unpublished observation). These experiments now need to be performed in the intact animal to test whether efficient gene transfer can be accomplished by this method. The major advantage of this technology is that the adenoviruses used in these types of experiments can be psoralen or UVinactivated, therefore reducing the possibility of viral gene expression, and potentially reducing the immune response (Cotten et al., 1992). If the immune response to adenovirus cannot be overcome, then nonviral substitutes must be developed for long-term efficient gene transfer. One promising report (Zhu et al., 1993) has shown that when mixed with cationic liposomes, DNA transfer into adult mice by intravenous injection is relatively efficient and expression can last for at least 9 weeks. The field of gene transfer is one that is receiving a great deal of attention, and substantial progress has been made in a relatively short time. Many of the areas in need of improvement or expansion have now been identified, and it seems clear that in the near future, safe and effective gene transfer will be in common use. References Ascadi, G., Jiao, S. S., Jani, A,, Duke, D., Williams, P., Chong, W., and Wolff, J. A. (1991).Direct gene transfer and expression into rat heart in vivo. New Biol. 3,71-81. Bett, A. J., Haddara, W., Prevec, L., and Graham, F. L. (1994). An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3. Proc. Nafl. Acad. Sci. USA 91,8802-8806. Buttrick, P. M., Kass, A., Kitsis, R. N., Kaplan, M. L., and Leinwand, L. A. (1992). Behavior of genes directly injected into rat heart in vivo. Circ.Res. 70, 193-198. Buttrick, P. M.,Kaplan, M. L., Kitsis, R. N., and Leinwand L. A. (1993). Distinct behavior of cardiac myosin heavy chain gene constructs in vivo-discordance with in vitro results. Circ. Res.
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Cotten, M., Wagner, E., Zatloukal, K.,Phillips, S., Curiel, D. T., and Birnstiel, M. L. (1992).Highefficiency receptor-mediated delivery of small and large (48 kilobase) gene constructs using the endosome-disruption activity of defective or chemically inactivated adenovirus particles. Proc. Natl. Acad. Sci. USA 89,6094-6098.
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Curiel,D. T., Agarwal,S., Wagner,E. and Cotten, M. (1991). A d e n o h enhancement of transferrinpolylysine-mediated gene delivery. Proc. NatL Acad. Sci. USA 88,8850-8854. Engelhardt, J. F., Ye, X.,Doranz, B., and Wilson, J. M. (1994). Ablation of E2A in recombinant adenoviruses improves transgene persistence and decreases inflammatory response in mouse liver. Proc. Natl. Acad. Sci. USA 91,61%-6200. Fynan, E. F., Webster, R. G., Fuller, D. H., Haynes, J. R., Santoro, J. C., and Robinson, H. L. (1993). DNA vaccines: Protective immunizations by parenteral, mucosal, and gene-gun innoculations. Proc. Natl. Acad. Sci USA 90,11478-11482. Hansen, E., Fernandes, K., Goldspink, G., Butterworth, P., Umeda, P. K., and Chang, K. C. (1991). Strong expression of foreign genes following direct injection into fish muscle. FEBS Letfers 290, 73-76. Jaffe, H. A., Danel, C., Longenecker, G., Metzger, M., Setouchi, Y., Rosenfeld, M. A., Gant, T. W., Thorgeirsson, S. S., Stratford-Pemcaudet, L. S., Pemcaudet, M., Pavirani, A., Lecocq, J.-P., and Crystal, R. G. (1992). Adenovims-mediated in vivo gene transfer and expression in normal rat liver. Nature Genetics 1,372-378. Kass-Eisler,A,, Falck-Pedersen,E., Alvira, M., Rivera, J., Buttrick, P. M., Wittenberg, B. A., Cipriani, L., and Leinwand, L. A. (1993). Quantitative determination of adenovirus-mediatedgene delivery to rat cardiac myocytes in vitro and in vivo. Proc. Natl. Acad. Sci. USA 90,11498-11502. Kass-Eisler, A., Falck-Pedersen, E., Elfenbein, D. H., Alvira, M., Buttrick, P. M., and Leinwand, L. A. (1994). Distribution, duration, and immune response to adenovirus-mediated gene transfer. Gene Therapy 1,395-402. Kitsis, R. N., Buttrick, P. M., McNally, E. M., Kaplan, M. L., and Leinwand L. A. (1991). Hormonal modulation of a gene injected into rat heart in vivo. Proc. Natl. Acad. Sci. USA 88,4138-4142. Kitsis, R. N., Buttrick, P. M., Kass, A. A., Kaplan, M. L., and Leinwand L. A. (1993). Gene transfer into adult rat heart in vivo. Methods in Molecular Genetics 1,374-392. Le Gal La Salle, G., Robert, J. J., Berrard, S., Ridoux, V.,Stratford-Perricaudet, L. D., Perricaudet, M., and Mallet, J. (1993). An adenovirus vector for gene transfer into neurons and glia in the brain. Science 259, 988-990. Lin, H., Parmacek, M.S., Morle, G., Bollling, S., and Leiden, J. M. (1990). Expression of recombinant genes in myocardium in vivo after direct injection of DNA. Circulation 82,2217-2221. Mittal, S . K., McDermott, M. R., Johnson, D. C., Prevec, L., and Graham, F. L. (1993). Monitoring foreign gene expression by a human adenovirus-based vector usning the firefly luciferase gene as a reporter. Virus Research 28,67-90. Quantin, B., Pemcaudet, L. D., Tajbakhsh, S., and Mandel, J-L. (1991). Adenovirus as an expression vector in muscle cells in vivo. Proc. Natl. Acad. Sci. USA 89,2581-2584. Rosenfeld, M. A., Siegfried, W., Yoshiura, K., Yoneyama, K., Fukayama, M., Stier, L. E., Paakko, P. K., Gilardi, P., Stratford-Pemcaudet, L. D., Pemcaudet, M., Jallat, S., Pavirani, A., Lecocq, J.-P., and Crystal, R. G. (1991). Adenovirus-mediated transfer of a recombinant al-antitrypsin gene to the lung epithelium in vivo. Science 252,431-434. Selden, R. F., Howie, K. B., Rowe, M. E., Goodman, H. M., and Moore, D. D. (1986). Human growth hormone as a reporter gene in regulation studies employing transient gene expression. Mol. Cell Biol. 6,3173-3179. Stratford-Pemcaudet, L. D., Makeh, I., Pemcaudet, M., and Briand, P. (1992). Widespread longterm gene transfer to mouse skeletal muscles and heart. J. Clin. Invest. 90, 626-630. Ulmer, J. B., Donelly, J. J., Parker, S. E., Rhodes, G. H., Felgner, P. L., Dwarki, V.J., Gromkowski, S. H., Deck, R. R., De Witt, C. M., Friedman, A., Hawe, L. A., Leander, K. R., Martinez, D., Perry, H. C., Shirer, J. W., Montgomery, D. C., and Liu, M. A. (1993). Heterologous protection against influenza by injection of DNA encoding a viral protein. Science 259,1745-1749. von Harsdorf, R., Schott, R. J., Shen, Y. T., Vatner, S. F., Mahdavi, V.,and Nadal-Ginard, B. (1993). Gene injection into canine myocardium as a useful model for studying gene expression in the heart of large mammals. Circ. Res. 72,688-695. Wolff, J. A., Malone, R. W., Williams, P., Chong, W., Ascadi, G., Jani, A., and Felgner, P. L. (1990). Direct gene transfer into mouse muscle in vivo. Science 247, 1465-1468.
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Wolff, J. A., Williams, P., Ascadi, G., Jiao, S.,Jani, A., and Chong, W. (1991). Conditions affecting direct gene transfer into rodent muscles in vivo. Biorechniques 11,474-485. Wolff, J. A., Ludtke, J. J., Acsadi, G., Williams, P., and Jani, A. (1992). Long-term persistence of plasmid DNA and foreign gene expression in mouse muscle. Human Molecular Genetics 1,363-369. Yang, Y.,Nunes, F. A., Berenni, K., Furth, E. E., Gonnol, E., and Wilson,J. M. (1994). Cellular immunity to viral antigens limits El-deleted adenoviruses for gene therapy. Proc. Natl. Acad. Sci. USA 91,4407-4411. Yoshimura, K., Rosenfeld, M. A., Seth, P., and Crystal, R. G. (1993). Adenovirus-mediated augmentation of cell transfection with unmodified plamid vectors. J. B i d Chem. 268,2300-2303. Zhu, N., Liggitt, D., Liu, Y.,and Debs, R. (1993). Systemic gene expression after intravenous DNA delivery into adult mice. Science 261,209-211.