In vivo gene transfer via intravenous administration of cationic ... - Nature

Gene Therapy (1997) 4, 891–900  1997 Stockton Press All rights reserved 0969-7128/97 $12.00

In vivo gene transfer via intravenous administration of cationic lipid–protamine–DNA (LPD) complexes S Li and L Huang Laboratory of Drug Targeting, Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA

A novel LPD formulation has been developed for in vivo gene transfer. It involves the interaction of plasmid DNA with protamine sulfate, a cationic polypeptide, followed by the addition of DOTAP cationic liposomes. Compared with DOTAP/DNA complexes, LPD offers better protection of plasmid DNA against enzymatic digestion and gives consistently higher gene expression in mice via tail vein injection. When a luciferase reporter gene was employed, gene expression was found in all tissues examined including lung, heart, spleen, liver and kidney with the highest expression in the lung. The in vivo efficiency of LPD was dependent upon charge ratio and was also affected by the lipid used. Increasing the amount of DNA delivered induced an increase in gene expression. The optimal dose was approximately 50 mg per mouse at which concentration approximately 20 ng luciferase protein per milligram extracted tissue protein could be detected in the lung.

Increasing the DNA to 100 mg per mouse resulted in toxicity and death of the animal. Gene expression in the lung was detected as early as 1 h after injection, peaked at 6 h and declined thereafter. High expression was also found in the spleen 6 h after injection but dropped very rapidly thereafter. The in vivo gene expression by LPD was dependent upon the route of administration since intraportal injection of LPD led to about a 100-fold decrease in gene expression in the lung as compared with i.v. injection. Using lacZ as a reporter gene, it was shown that endothelial cells were the primary locus of transgene expression in both the lung and spleen. No sign of inflammation in these organs was noticed. Since protamine sulfate has been proven to be nontoxic and only weakly immunogenic in humans, this novel vector may be useful for clinical gene therapy.

Keywords: gene transfer; liposomes; polymer; plasmid DNA; gene therapy

Introduction Gene therapy could represent an important advance in the treatment of both inherited and acquired diseases.1,2 Success of human gene therapy depends upon the development of delivery vehicles or vectors which can selectively deliver therapeutic genes to target cells with efficiency and safety. Viral vectors, although highly efficient, suffer from a number of problems such as immunogenicity,3 toxicity4 and potential recombination or complementation.5 As a result of these limitations, much effort has been devoted to the development of nonviral vectors, such as cationic liposomes6,7 and cationic polymers.8,9 Cationic liposomes are particularly attractive due to their favorable characteristics such as biodegradability, minimal toxicity, nonimmunogenicity, relative ease of large-scale production and simplicity of use. Since the description of the first cationic lipid by Felgner in 1987,10 numerous new lipids have been reported. 6,7 Currently, cationic liposomes are widely employed for the transfection of eukaryotic cells in research laboratories. Several liposomal formulations have also undergone clinical evaluation as vectors for gene therapy in cancer and cystic fibrosis. 11–13

Correspondence: L Huang Received 17 March 1997; accepted 9 May 1997

While early laboratory studies and clinical trials have demonstrated the potential of cationic liposomes in gene therapy, they have also revealed the insufficient activity of gene transfer of the first generation cationic liposomes.14 Thus, substantial effort has been spent towards improving their efficiency. These are largely based on our better understanding of the structure/function relationship of the cationic lipids.15–17 For example, transfection efficiency can be improved significantly by designing and synthesizing new lipids.17,18 Cationic lipid–DNA complexes can also be prepared in such a way that they are highly efficient and serum resistant.19–22 Another direction of research aimed at improving the efficiency of lipofection is based on the effort to condense DNA in a manner similar to natural vectors such as virus and sperm. These studies are aimed at developing a nonviral, selfassembling system, or ‘artificial virus’.23 Recent study in our laboratory has led to the development of a novel formulation, ie liposome–polycation–DNA complexes (LPD).24 It involves the use of a cationic polymer in addition to a cationic lipid to condense DNA. Interaction of these components at an appropriate ratio results in the formation of a condensed DNA core coated with a lipidic shell, resembling the structure of a virus. This artificial virus is more efficient than the first generation cationic liposomes in transfecting cells in vitro24 and also in transfecting brain tissue in vivo (During et al, unpublished data). In this study, we report that LPD, when

In vivo gene transfer by LPD S Li and L Huang

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appropriately formulated, gives high gene expression in vivo, particularly in the lung, following intravenous administration.

Results In vivo gene expression of LPD is charge ratio (+/−) dependent The charge ratio of either LPD or DOTAP–DNA complexes is calculated based on the assumptions that 1 nmol of DOTAP and protamine sulfate contribute 1 and 21 nmols of positive charge, respectively, and that 1 mg DNA has 3.1 nmols of negative charge. LPD was prepared by mixing protamine with DNA at a +/− charge ratio of 1:1 followed by the addition of varying concentrations of DOTAP liposomes. The size of the resulting complexes ranged from 200 to 300 nm in diameter. Figure 1 shows the in vivo gene expression of LPD as a function of DOTAP concentration. The amount of DNA used was 50 mg per mouse. It can be seen that gene expression was found in all tissues examined, including heart, lung, liver, spleen and kidney, with the highest expression found in the lung. Increasing the amount of DOTAP resulted in a steady increase in gene expression in the lung and spleen; however, the extent of charge ratio dependence was closely related to the dose of DNA. The charge ratio dependence was more striking when a low dose of DNA was used and became less obvious with increasing amounts of DNA (data not shown). The requirement of high +/− charge ratio for efficient in vivo gene transfer seems to be a general phenomenon for cationic lipids as a similar pattern was also found when other cationic lipids were used, including 3-b-[N-(N′-N′-dimethyl-ethane)carbamoyl]cholesterol (DC-chol), 3-b-[N-(N′-N′-tri-

Figure 1 In vivo gene expression of LPD as a function of DOTAP concentration. pCMVL DNA was mixed with protamine (0.8 mg protamine per microgram DNA) followed by the addition of various amounts of DOTAP liposomes. Dextrose was added to the mixture to a final concentration of 5%, and 50 mg of pCMVL DNA was injected intravenously into each mouse (CD-1 female; 4–6 weeks old). Twenty-four hours following the injection, mice were killed and major organs were collected. Tissues were homogenized in lysis buffer. The samples were centrifuged at 14 000 g for 10 min, and the supernatant was assayed for luciferase activity and protein concentration, respectively. The results are expressed as nanograms of luciferase per milligram of protein (n = 3).

methyl-ethane)-carbamoyl]-cholesterol (TC-chol) and dimethyl-dioctadecyl-ammonium bromide (DDAB). However, the efficiency of gene expression differed greatly among these lipids whether as lipid–DNA complexes or as LPD with the highest gene expression found with DOTAP. The in vivo gene expression of LPD was also increased with increasing amounts of protamine. However, LPD became unstable and tended to form large aggregates when the charge ratio of protamine–DNA was greater than 2:1. Therefore, a 1:1 charge ratio of protamine–DNA was employed for LPD in all subsequent studies.

Dose effect and duration of gene expression Figure 2 depicts the gene expression of LPD as a function of DNA concentration. The lipid:DNA charge ratio was 11:1 for all doses and the injection volume was 300 ml. Increasing the amount of DNA led to a steady increase in gene expression in all the tissues examined, except for the spleen in which gene expression was saturated when the dose reached 50 mg per mouse. The lung remained the tissue with the highest level of gene expression at all doses tested. Gene expression in the heart was increased significantly when the DNA dose was increased to 75 mg per mouse. One of the three mice died of toxicity when using 100 mg of DNA per mouse, yet no signs of toxicity were noticed when the dose used was 75 mg DNA per mouse or below. Figure 3 shows the duration of gene expression by LPD. The amount of DNA injected was 50 mg per mouse and the lipid:DNA charge ratio was 11:1. Transgene expression was detected as early as 1 h following i.v. injection of LPD, peaked at 6 h and declined thereafter. Gene expression in the lung was significantly higher than that found in other tissues at all time-points except for the spleen at early time-points. Six hours after i.v. injection of LPD, gene expression in the spleen reached a level comparable with that of the lung, but declined very rapidly thereafter. In contrast, a relatively high level of

Figure 2 In vivo gene expression of LPD as a function of DNA concentration. LPD was prepared at a protamine:DNA:DOTAP charge ratio of 1:1:11. Different amounts of DNA were injected into mice and gene expression was assayed 24 h later as described in the legend to Figure 1 (n = 3).


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Figure 3 In vivo gene expression of LPD as a function of time. LPD was prepared as described in the legend to Figure 2 and injected into the mice at 50 mg DNA per animal. At different times following the injection, mice were killed and major organs were assayed for gene expression (n = 3).

expression lasted up to 2 days in the lung. Four days after injection of LPD, gene expression could still be detected in the lung, spleen and liver.

Sequential injection of protamine–DNA complexes and free DOTAP liposomes While a high DOTAP:DNA molar ratio is essential for the efficient in vivo gene transfer by LPD, it is conceivable that significant amounts of DOTAP liposomes remain free in the LPD preparations. To better understand how the free cationic liposomes help to improve the in vivo performance of LPD, protamine–DNA complexes and free DOTAP liposomes were i.v. administered separately at intervals of 3 min and 15 min, respectively, and gene expression in major organs was determined 24 h later. Protamine–DNA complexes were preformed at a +/− charge ratio of 1:1 and the amount of DNA was 50 mg per mouse. The amount of DOTAP liposomes used per mouse was 1.16 mg, the same amount as when DOTAP liposomes were utilized to prepare LPD. In one experiment, protamine–DNA complexes were injected first, followed by the injection of DOTAP liposomes. In another experiment, DOTAP liposomes were administered first, followed by the injection of protamine–DNA complexes. The results of both experiments are shown in Figure 4. When the duration of the interval between the two injections was 3 min, gene expression in the lung was reduced by three- to five-fold as compared with co-injection, ie the regular LPD protocol. No significant differences were found with respect to the order of which component was injected first. Prolonging the interval between the two injections from 3 to 15 min further decreased the level of gene expression in vivo, especially when protamine–DNA complexes were administered first. Only minimal activity

Figure 4 Sequential injections of protamine–DNA complexes and free DOTAP liposomes and their in vivo gene expression. Protamine–DNA complexes were prepared at a +/− charge ratio of 1:1 and DOTAP liposomes were prepared by sonication. Groups of three mice received i.v. injection of protamine–DNA complexes (50 mg per mouse) first, followed by free DOTAP liposomes (1.16 mg) at intervals of 3 min and 15 min, respectively (a). In a separate experiment (b), mice received injection of free DOTAP liposomes first, followed by protamine–DNA complexes (PD). Gene expression was assayed 24 h after the injection as described in the legend to Figure 1 (n = 3).

of transgene product could be detected in the spleen. Reversing the sequence of the two injections significantly increased gene expression in the lung. The level of expression, however, varied greatly among different animals of the same experiment and also differed significantly from experiment to experiment (n = 4) (data not shown). No difference in toxicity was found whether animals were treated with LPD or received separate injections of protamine–DNA complexes and DOTAP liposomes.


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In vivo distribution of DNA and lipid following i.v. injection of LPD To understand further how an excess amount of lipid affects the in vivo fate of protamine–DNA, DNA was labeled with 125I and the liposomes were labeled with 111In and distribution of DNA and lipid in the liver and lung were followed after injection of protamine–DNA complexes, DOTAP liposomes or LPD. The results are shown in Figure 5. DNA was rapidly removed from circulation by the liver after injection of protamine–DNA complexes, the uptake of DNA by the lung was below 10% of injected dose at all time-points examined. When DNA was formulated in LPD, substantial amounts were trapped in the lung after i.v. injection. Five minutes after injection, about 40% of the injected dose was found in the lung. It was then slowly released from the lung with time and redistributed to the liver. 111In followed a similar distribution pattern as that of 125 I except that its recovery in the liver was higher than that of 125I . This might be due to the degradation of 125I-labeled DNA in the liver. Injection of DOTAP alone showed a tissue distribution similar to that of DOTAP liposomes injected together with protamine–DNA complexes; however, their residence in the lung did not last long (data not shown). Effect of administration routes on the in vivo efficiency of LPD To determine the effect of administration routes on in vivo performance, LPD was also injected intraportally and in vivo gene expression was compared with that of intravenous injection. As shown in Figure 6, gene expression in the lung was reduced dramatically when LPD was injected intraportally. However, gene expression in the lung was still higher than that of the

Figure 5 In vivo distribution of protamine–DNA complexes or LPD. DNA was labeled with 125 I and LPD and protamine–DNA complexes were prepared as described in the legend to Figure 1. Groups of three mice received i.v. injection of protamine–DNA complexes or LPD. At different times following the administration, mice were bled and killed by cervical dislocation. Major organs were removed and assayed for radioactivity. The uptake of 125 I by each organ was expressed as the percentage of injected dose. (P), protamine–DNA by lung; (p), protamine–DNA by liver; (G), LPD by lung; (g), LPD by liver.

Figure 6 Effect of administration routes on the in vivo gene expression by LPD. Groups of three mice received LPD at a dose of 50 mg DNA per mouse intravenously or intraportally and gene expression was assayed 24 h after injection. LPD was prepared as described in the legend to Figure 2.

liver. Gene expression was also detected in the spleen upon intraportal injection of LPD.

Southern blot analysis of plasmid DNA extracted from the tissues of injected mice The rapid decline of gene expression in vivo might be due to the rapid clearance from the tissues and/or degradation within the tissues of the injected DNA. To test this hypothesis, groups of three mice were injected with LPD, DOTAP–DNA complexes and protamine–DNA complexes, respectively, and the lung and spleen were collected at different time-points following the injection. DNA was extracted from the tissues and subjected to Southern blot analysis using luciferase cDNA as a probe. The result is shown in Figure 7. Plasmid DNA was found in the lungs of mice treated with LPD or DOTAP–DNA complexes 1 and 6 h after i.v. administration but became barely detectable at 24 h. The amount of DNA in LPDtreated mice was higher than those in DOTAP–DNAtreated mice at all time-points examined. No injected plasmid DNA was detected in the lungs in protamine– DNA-treated group at any time-point (Figure 7a). Analysis of DNA extracted from the spleen exhibited a similar result (Figure 7b) even though gene expression in the spleen dropped much faster. In vitro DNaseI protection assay To confirm further the role of protamine in protecting the DNA from attack by degrading enzymes in vivo, an in vitro DNaseI protection assay was performed. Plasmid DNA was complexed with DOTAP, protamine, or formulated in LPD. By varying the amount of DOTAP, LPD or DOTAP–DNA of different +/− charge ratios were prepared. The result is shown in Figure 8. At a +/− charge ratio of 4:1, DOTAP offered partial protection for the DNA. In contrast, the DNA was completely protected from the digestion by DNaseI when formulated in LPD of the same charge ratio. At a +/− charge ratio of 12:1, no sign of DNA degradation was found for both DOTAP–DNA complexes and LPD. Protamine alone only provided partial protection for DNA at a charge ratio of 1:1, while the DNA was poorly protected when complexed with DOTAP at the same charge ratio.


Figure 7 Southern analysis of the plasmid DNA extracted from tissues of injected mice. Groups of three mice received i.v. injection of protamine– DNA complexes, DOTAP–DNA complexes or LPD at a dose of 50 mg per animal. At indicated times after injection, lung and spleen were collected. Total DNA was extracted from the tissues and subjected to analysis using 32P-labeled luciferase cDNA as a probe.

Figure 8 In vitro DNaseI protection assay. LPD, protamine–DNA complexes or DOTAP–DNA complexes of different charge ratios were prepared. DNaseI was added to each sample to a final concentration of 1 U/mg DNA, and the mixtures were incubated at 37°C for 30 min. SDS was added to each sample to a final concentration of 1%, and the treated samples were analyzed on a 0.8% agarose gel using untreated plasmid DNA as a control. A and I, control plasmid; B, plasmid alone; C, liposome–DNA (+/−, 12/1); D, liposome–DNA (+/−, 4/1); E, liposome–DNA (+/−, 1/1); F, LPD (+/−, 12/1); G, LPD (+/−, 4/1); H, PD (+/−, 1/1).

Identification of transfected cell type Having defined the optimal conditions with pCMVL, in vivo gene transfer by LPD was further evaluated using pCMVLacZ as a reporter gene. Mice received intravenous injection of LPD containing lacZ DNA with either a human CMV promoter or promoterless expression system. Twenty-four hours after injection, mice were killed and lungs were fixed and stained for b-galactosidase activity using X-gal at 37°C. Figure 9 depicts the appearance of lungs viewed under a dissecting microscope. Homogeneous expression was found in lobes (Figure 9a) of the mice treated with LPD containing pCMVLacZ. No expression was detected in the lungs of mice treated with promoterless plasmid (Figure 9b). Similar results were found when the tissue sections of the lobes in Figure 9 were examined under light microscope. No blue cells were observed in the lung treated with promoterless plasmid (Figure 10b). In contrast there was localized gene expression throughout the distal lung (Figure 10a) of mouse treated with LPD containing pCMVLacZ. At higher magnification, the primary loci of lacZ expression appeared to be capillary endothelium located within the alveolar septum (Figure 10d). Endothelial cells were also found to be the cell type transfected in the spleen (data not shown). No sign of inflammation was noticed in all tissues examined. Comparison of several polymers for their capacity to form an efficient LPD Recent studies in our laboratory have demonstrated that polylysine and protamine of different salt forms vary in their ability to enhance lipofection in vitro.37 In this study, protamine-free base, protamine phosphate and poly(llysine) hydrobromide were also used to prepare LPD and their in vivo transfection efficiency was compared with that of LPD containing protamine sulfate-USP (Figure 11). Consistent with what was found in vitro, protamine sulfate-USP was also the most efficient in enhancing in vivo lipofection. LPD prepared with protamine-free base was not stable and tended to form aggregates. Figure 11 also shows that the protamine sulfate–DNA complexes without lipid were inactive. Protamine-free base–DNA complexes and protamine phosphate–DNA complexes were also not active (data not shown). Naked DNA alone did not give any gene expression in vivo. DOTAP–DNA complexes were effective in transfecting cells in vivo, however, the expression level of the complexes varied from experiment to experiment (the data shown in Figure 11 represent one of the successful experiments). Inclusion of protamine into DOTAP–DNA complexes gave consistently higher gene expression in vivo, particularly in the lung. Normally, inclusion of protamine increased the gene expression of DOTAP–DNA by five- to 10-fold in the lung and 10- to 50-fold in the liver or spleen. Inclusion of protamine was also found to enhance the efficiency of gene delivery by other liposomal formulations including DC-chol, TC-chol and DDAB.

Discussion Previous work by Gao and Huang has shown that inclusion of a cationic polymer into cationic lipid–DNA complexes enhances the efficiency of lipofection in vitro.24 Possible mechanisms for this enhancement include improved protection of DNA against enzyme attack,

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Figure 9 b-Galactosidase staining of lungs 24 h after i.v. injection of LPD containing lacZ cDNA with either a human CMV promoter (a) or promoterless expression system (b). The lung processing and staining were performed as described in Materials and methods, and examined with a dissecting microscope at × 4. Homogeneous expression was apparent in the lobe (a) isolated from mice treated with LPD containing plasmid expression vector with a human CMV promoter; there was no detectable transgene expression in the lung after injection of LPD containing promoterless expression vector (b).

Figure 10 Light photomicrograph of lung sections of the mice injected with LPD containing lacZ cDNA (with a hCMV promoter). Separate lung lobes from mice described in the legend to Figure 9 were embedded in paraffin and thin sections were counterstained and viewed with a Nikon light microscope (Nikon, Garden City, NY, USA) at × 20 (a and b) and × 100 (c and d). There was no detectable lacZ expression in the lung of control mouse (b). In contrast, there was localized expression throughout the distal lung (a, arrows) of mouse injected with LPD containing lacZ with a hCMV promoter. At higher magnification (c and d), expression was seen in structures within the alveolar septum that appeared to be capillary endothelium as evidenced by appearance of X-gal product at both surfaces of the capillary endothelium (double arrows, d).

assistance afforded by the polymer in the nuclear transport of plasmid DNA, and more importantly, more efficient uptake by cells of the ternary complexes owing to the favorable structure of the highly condensed, lipidassociated particles. Different polymers or even the same polymer of different molecular weights vary in their

ability to enhance lipofection. A polylysine with a molecular weight of 25 600 was found to be the best among several polymers examined.24 As part of a continuing effort to improve further the efficiency of LPD, protamine-sulfate was subsequently discovered to be more efficient than polylysine in enhancing lipofection.37


Figure 11 Comparison of several different formulations for their in vivo gene expression efficiency. LPD containing polylysine hydrobromide (1), protamine-free base (2), protamine phosphate (3), and protamine sulfateUSP (4), respectively, was prepared as described in the legend to Figure 2. PD, protamine sulfate–DNA complexes; LD, DOTAP cationic liposome– DNA complexes. The effect of naked DNA is also shown (n = 3).

It is interesting, however, to find that protamine phosphate or protamine-free base worked poorly in enhancing lipofection. The reason for the discrepancy among protamine of different salt forms remains unknown. Yet, the data clearly suggest that protamine sulfate is a better candidate for the preparation of LPD. There are additional advantages of utilizing protamine sulfate and these are related to its biocompatibility. Protamine sulfate-USP is nontoxic and only weakly immunogenic in humans and has a record of wide clinical application. Recently, LPD containing protamine sulfate-USP has been shown to transfect brain tissue efficiently and it is currently being used in a clinical trial for the treatment of Canavan’s disease (During et al, unpublished data). In this study, we further examine the in vivo gene expression by this novel LPD upon i.v. administration. As shown in Figure 11, DOTAP–DNA complexes produced some gene expression in the lung; however, the expression level of the complexes varied greatly from experiment to experiment. This might be due to some subtle difference in the structure of complexes from batch to batch. It is generally known that cationic lipid–DNA complexes are very heterogeneous in structure.25,26 Different populations of the complexes may differ greatly in their sensitivity to serum, uptake by cells and eventually intracellular trafficking and gene expression. The way the DNA is mixed with liposomes affects the efficiency of the resulting complexes greatly.20 It has been shown in a recent study that sequential addition of cationic lipid to DNA results in formation of complexes which are not only more efficient but also serum resistant. The starting concentration of either DNA or liposomes also significantly affects the lipofection efficiency.20 Therefore, preparation of cationic liposome–DNA complexes is a process that is difficult to control, which might explain the large variation for the in vivo lipofection by DOTAP liposomes. Cationic polymers behave differently from cationic liposomes in their interaction with DNA. They interact with DNA in a more controllable manner which is less sensitive to the above-mentioned factors.20 This might explain

the more consistent in vivo lipofection result when LPD was used. At present, the detailed mechanisms for the improved in vivo lipofection by LPD are not fully understood. One likely mechanism is improved protection of the DNA by LPD as compared with cationic liposomes. This was confirmed in a Southern blot of the DNA extracted from tissues of treated mice (Figure 7) and DNaseI protection assay (Figure 8). LPD was more efficient than DOTAP in protecting the DNA from attack by both DNaseI and DNA degrading enzymes in vivo. Interesting to note is that polylysine was more efficient than protamine in protecting the DNA from digestion by DNaseI (data not shown) but less active in enhancing in vivo lipofection. Obviously, other factors may also be important in determining the capacity of a cationic polymer to form an efficient LPD. It should be noted that polylysine was used in this study according to conditions optimized for protamine sulfate. More studies of polylysine are therefore needed before any definite conclusion can be made. It is generally known that lipofection is sensitive to serum.27 A recent study in this laboratory has demonstrated that serum sensitivity of in vitro lipofection can be overcome by increasing the charge ratio of cationic lipid–DNA.38 This study also demonstrates that a high +/− charge ratio is essential for in vivo gene transfer by LPD (Figure 1). A similar result has been found in a separate study by Liu et al28 using DOTMA/Tween 80 for intravenous delivery of DNA. It has been shown recently by Xu and Szoka29 that DNA can be released from cationic liposome–DNA complexes by various anionic molecules in vitro, rendering the DNA more susceptible to digestion by DNase I. It was speculated that DNA could be similarly released from lipid–DNA complexes in vivo, a barrier for cationic lipid-mediated gene transfer.29 The presence of an excess amount of cationic lipid may help neutralize hostile factors in the serum and therefore protect the integrity of protamine–DNA or LPD. This was supported by our Southern blot analysis of the DNA extracted from tissues of treated mice: much more DNA was detected in the mice treated with LPD as compared with that in the mice treated with protamine–DNA complexes (Figure 7). DOTAP liposomes can also prolong the residence of intact DNA in tissues particularly in the lung as shown in the biodistribution study (Figure 5). Taken together, these results provide for more efficient interaction of intact DNA with target cells (endothelial cells), resulting in a high level of gene expression in vivo. The protective role of excess lipids, however, was effectively manifested only when they were co-injected with protamine–DNA complexes, ie the regular LPD protocol. Separate administration of protamine–DNA and cationic liposomes resulted in a significant reduction in gene expression, especially when the interval between the two injections was prolonged (Figure 4). It might be possible that lipid and protamine–DNA interact with each other in the presence of serum to form a new structure which is efficient in transfecting endothelial cells. Sequential injections of free liposomes and protamine–DNA might decrease the chance of interaction between the two components resulting in a decrease in gene expression. Currently, it is not known how free liposomes interact with protamine–DNA complexes or with preformed LPD in vivo. Understanding this process might help to clarify

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the mechanism of in vivo lipofection and design better formulations for intravenous applications. While we were preparing this manuscript, several other liposome–DNA complexes were reported to give a high level of gene expression in the lung after i.v. administration.30,31 The activity varied with the cationic lipids employed, the size of liposomes and the helper lipids used. When the same liposomes were used to prepare LPD, changes in each of those parameters were also found to influence the in vivo activity of LPD. Yet, inclusion of protamine consistently increased the in vivo activity of liposome–DNA complexes. More importantly, LPD was more stable upon storage and gave less toxicity as compared with liposome–DNA complexes (Li and Huang, unpublished data). In conclusion, a novel LPD composed of DOTAP, protamine and DNA has been developed in this study which is highly efficient for in vivo gene transfer. The in vivo performance of LPD is charge ratio-dependent and also dose-dependent. Preliminary studies have shown that the LPD is quite stable and can be stored at 4°C for 4 weeks without losing its activity. Future studies are to search for more potent cationic lipids and/or to incorporate into LPD a component(s) which could further improve its in vivo transfection efficiency.

Materials and methods Chemicals 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) was purchased from Avanti Lipid (Alabaster, AL, USA). Poly(l-lysine) (PLL) hydrobromide (MW 25 600), protamine-free base and protamine phosphate were supplied by Sigma (St Louis, MO, USA). Protamine sulfateUSP was from Eli Lilly (Indianapolis, IN, USA). Luciferase assay kit was obtained from Promega (Madison, WI, USA). All other chemicals were of reagent grade. DOTAP liposomes were prepared in filtered distilled water by sonication with a final concentration of 50 mg lipid/ml. Plasmids Plasmids pCMVL and pCMVLacZ, which contain, respectively, the cDNA of firefly luciferase and bacterial b-galactosidase (b-gal) driven by a human cytomegalovirus immediate–early promoter, were amplified in DH5a strain of E coli, isolated by alkaline lysis and purified by cesium chloride centrifugation.32 Preparation of LPD and DOTAP–DNA complexes For preparation of LPD, DNA was diluted in distilled water and mixed with diluted protamine at a weight ratio of 0.8 mg of protamine per microgram of DNA. The mixture was allowed to stand at room temperature for 10 min. Different amounts of DOTAP liposomes were added to the solution and the mixture was incubated at room temperature for another 10 min before use. LPD containing polylysine was similarly prepared. For preparation of DOTAP–DNA complexes, DOTAP liposomes were added to the diluted DNA in distilled water and the mixture was incubated at room temperature for 10 min before use. The size of DOTAP–DNA complexes and LPD was determined by dynamic laser light scattering using a Coulter N4SD particle sizer (Hialeah, FL, USA).

Injection of LPD Female CD-1 mice of 4–6 weeks of age were employed. They were purchased from Charles River Laboratories (Wilmington, MA, USA) and were housed in accordance with institutional guidelines. Individual mice in groups of three were injected intravenously with 50 mg of DNA complexed with DOTAP alone, protamine alone or formulated in LPD in a total volume of 300 ml 5% w/v glucose. Twenty-four hours following i.v. injection, mice were killed and major organs were removed and assayed for gene expression. For intraportal injection, mice were anesthetized with inhalation of methoxyflurane (Mallinckrodt Veterinary, Mundelein, IL, USA) and intramuscular injection of ketamine hydrochloride (1 mg/20 g body weight). After the portal vein was exposed, 50 mg of DNA formulated in LPD was administered using a tuberculin syringe with a 30-gauge .-inch needle. Gene expression in major organs was assayed 24 h following the injection. Assay for luciferase activity The mice were bled from the retro-orbital sinuses under anesthesia and were then killed by cervical dislocation. Heart, lung, spleen, liver and kidney were collected and washed with cold saline twice. The organs were homogenized with lysis buffer (0.05% Triton X-100, 2 mm EDTA, 0.1 m Tris, pH 7.8) using a tissue tearor (Biospec Products, Bartlesville, OK, USA). After two cycles of freeze and thaw, the homogenates were centrifuged at 14 000 g for 10 min at 4°C and 20 ml of the supernatant was analyzed for luciferase activity using an Automated LB 953 luminometer equipped with an automated injector (Berthold, Bad Wildbad, Germany). Relative light units (RLU) were converted to protein (pg) of luciferase using purified enzyme (Calbiochem-Novabiochem, La Jolla, CA, USA) as a standard. Conversion was calculated according to: luciferase (pg) = (8.3 × 10−3 × RLU) − 5 (r2 = 0.99). X-gal staining Twenty four hours following the i.v. injection of pCMVLacZ formulated in LPD, the mice were killed and lungs were perfused intravascularly with 2% paraformaldehyde and 0.1% glutaraldehyde and inflated with this mixture to near total lung capacity. After rinsing with cold PBS, the lungs were incubated in a staining solution (0.08% 5-bromo-4-chloro-3-indolyl-b-d-galactoside, 5 mm of K3 Fe(CN)6, 2 mm MgCl2 in PBS) at 37°C for 24 h. The lungs were then embedded in paraffin and thin sections were prepared. The sections were counterstained with hematoxylin eosin. In vivo distribution of LPD Plasmid DNA was labeled with 125I by using a published method.33 DOTAP liposomes were labeled with 111Indiethylenetriamine penta-acetic acid distearylamide complex (DTPA-SA) as described.34 Radiolabeled protamine– DNA complexes and LPD were prepared the same way as described above. Groups of three mice received i.v. injections of DOTAP liposomes, protamine–DNA complexes or LPD. At different times following the injection, the mice were bled and killed by cervical dislocation. Major organs were collected and assayed for radioactivity. For dual-label studies, channel windows were set to collect 111In and 125I c.p.m. individually, and cross-talk


was accounted for appropriately. The total radioactivity in blood was determined by assuming the total blood volume was 7.3% of the body weight.35 The result was expressed as the percentage of injected dose per organ.

Determination of luciferase DNA in tissues by Southern blot analysis Fifty micrograms of pCMVL complexed with protamine, DOTAP liposomes or formulated in LPD was intravenously injected into each mouse. At different times after the injection, mice were killed and the lungs and spleen were collected. The tissues were washed twice with ice cold PBS, cut into pieces and were frozen in liquid nitrogen. The frozen tissues were crushed with a prechilled hammer to a fine powder and were then added into a digestion buffer (100 mm NaCl, 10 mm TrisCl, pH 8, 25 mm EDTA, 0.5% sodium dodecyl sulfate, 20 mg/ml RNase A and 0.1 mg/ml proteinase K). The samples were incubated with shaking at 50°C for 12 h. Digested samples were extracted twice with phenol/chloroform/isoamyl alcohol. DNA was precipitated with ethanol and resuspended in TE. Two hundred nanograms of extracted DNA was digested with HindIII for 1 h, run on 1% TAE agarose gels and blotted on to Nytran Nylon membranes (Schleicher and Schuell, Keene, NH, USA). The blots were prehybridized and hybridized as described.36 A gel-purified (Bio 101, La Jolla, CA, USA) fragment of the whole luciferase cDNA was radiolabeled using a nick translation system (Promega, Madison, WI, USA) and used as a probe. DNase I protection assay LPD, protamine–DNA complexes and liposome–DNA complexes were prepared as described above. Into 5 mg (DNA) of each sample was added DNase I to a final concentration of 1 U/mg DNA and the mixtures were incubated at 37°C for 90 min. SDS was added to the samples to a final concentration of 1% to release DNA from cationic liposomes and protamine. Samples were then analyzed by agarose gel electrophoresis and the integrity of DNA in each formulation was compared with untreated plasmid DNA as a control.

Acknowledgements The work was supported by NIH grants CA 59327, DK 44935, CA 64654, and a contract from Targeted Genetics Corporation. We thank Drs Bruce Pitt and Simon Watkins for assistance in the lacZ experiment described in Figures 9 and 10, and Dr Frank Sorgi for his valuable suggestion of using protamine sulfate for this work.

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