Optimization Of Targeted Delivery

Much effort has been made to specifically deliver nucleic acid-liposome complexes to target organs, tissues, and/or cells. Ligands that bind to cell surface receptors are usually attached to PEG and then attached to the cationic or anionic delivery vehicle. Due to shielding the positive charge of ca-tionic complexes by PEG, delivery to the specific cell surface receptor can be accomplished by only a small fraction of complexes injected systemically. Furthermore, delivery of PEGy-lated complexes into the cell occurs predominantly through the endocytic pathway, and subsequent degradation of the bulk of the nucleic acid occurs in the lysosomes. Thus, gene expression is generally lower in the target cell than using the nonspecific delivery of highly efficient cationic complexes.

As discussed above, the vast majority of the injected PEG-ylated complexes bypasses the target cell. Apparently, the PEGylated complexes cannot use critical charge interactions for optimal transfection into cells by direct fusion. Inability to expose positive charge on the surface of optimized complexes results in the transfection of fewer cells. PEGylation was first used to increase the half-life of complexes in the circulation and to avoid uptake in the lung. However, this technology also destroys the ability to efficiently transfect cells. We were able to increase the half-life in circulation of BIVs to 5 h without the use of PEG. Because the extended half-life of BIVs is not too long, this delivery system does not result in the accumulation of complexes in nontarget tissues that circulate for 1 to 3 days. Some investigators have now reported targeted delivery that produces increased gene expression in the target cell over their nontargeted complexes. However, these nontargeted and targeted delivery systems are inefficient (37) compared with efficient delivery systems such as the BIVs.

In using the extruded BIV DOTAP:Chol nucleic acid:lipo-some complexes, we produced an optimal half-life in the circulation without the use of PEG (14). Extended half-life was produced primarily by the formulation, preparation method, injection of optimal colloidal suspensions, serum stability, and optimal nucleic acid:lipid ratio used for mixing complexes, and size (200-450 nm). Furthermore, we avoid uptake in the lungs using the negative charge of the ligands and ''shielding/ deshielding compounds'' that can be added to the complexes used for targeting just prior to injection or administration in vivo. Our strategy to bypass nonspecific transfection is called reversible masking. By adding ligands using the novel approaches that we developed, adequate overall positive charge on the surface of complexes is preserved. In summary, we achieve optimal circulation time of the complexes, reach and deliver to the target organ, avoid uptake in nontarget tissues, and efficiently interact with the cell surface to produce optimal transfection.

XI. EFFICIENT DISSEMINATION THROUGHOUT TARGET TISSUES AND MIGRATION ACROSS TIGHT BARRIERS

A primary goal for efficient in vivo delivery is to achieve extravasation into and penetration throughout the target organ/ tissue ideally by noninvasive systemic administration. Without these events therapeutic efficacy is highly compromised for any treatment including gene and drug therapies. Achieving this goal is difficult due to the many tight barriers that exist in animals and people. Furthermore, many of these barriers become tighter in the transition from neonates to becoming adults. Penetration throughout an entire tumor is further hindered due to the increased interstitial pressure within most tumors (38-40). We believe that nonviral systems can play a pivotal role in achieving target organ extravasation and penetration needed to treat or cure certain diseases. Our preliminary studies have shown that extruded BIV DOTAP:Chol nucleic acid:liposome complexes can extravasate across tight barriers and penetrate evenly throughout entire target organs, and viral vectors cannot cross identical barriers. These barriers include the endothelial cell barrier in a normal mouse, the posterior blood retinal barrier in adult mouse eyes, complete and even diffusion throughout large tumors (15), and penetration through several tight layers of smooth muscle cells in the arteries of pigs (35). Diffusion throughout large tumors was measured by expression of p-galactosidase or the proapoptotic gene p53 in about half of the p53-null tumor cells after a single injection of BIV DOTAP:Chol-DNA liposome complexes into the center of a tumor. Transfected cells were evenly spread throughout the tumors. Tumors injected with complexes encapsulating plasmid DNA encoding p53 showed apoptosis in almost all the tumor cells by TUNEL staining. Tumor cells expressing p53 mediate a bystander effect on neighboring cells perhaps due to up-regulation by Fas ligand that causes nontransfected tumor cells to undergo apoptosis. Currently, we are investigating the mechanisms used by extruded DOTAP:Chol nucleic acid:liposome complexes to cross-barriers and penetrate throughout target organs. By knowing more about these mechanisms, we hope to develop more robust nonviral gene therapeutics.

XII. OPTIMIZATION OF PLASMIDS FOR IN VIVO GENE EXPRESSION

Delivery of DNA and subsequent gene expression may be poorly correlated (18,41). Investigators may focus solely on the delivery formulation as the source of poor gene expression. In many cases, however, the delivery of DNA into the nucleus of a particular cell type may be efficient, although little or no gene expression is achieved. The causes of poor gene expression can be numerous. The following issues should be considered independent of the delivery formulation, including suboptimal promoter enhancers in the plasmid, poor preparation of plasmid DNA, and insensitive detection of gene expression.

Plasmid expression cassettes typically have not been optimized for animal studies. For example, many plasmids lack a full-length cytomegalovirus (CMV) promoter enhancer. Over 100 variations of the CMV promoter enhancer exist, and some variations produce greatly reduced or no gene expression in certain cell types (18). Even commercially available plasmids contain suboptimal CMV promoters enhancers, although these plasmids are advertised for use in animals. Furthermore, upon checking the company data for these plasmids, one would discover that these plasmids have never been tested in animals and have been tested in only 1 or 2 cultured cell lines. Conversely, plasmids that have been optimized for overall efficiency in animals may not be best for transfection of certain cell types in vitro or in vivo. For example, many investigators have shown that optimal CMV promoters enhancers produce gene expression at levels several orders of magnitude less in certain cell types. In addition, one cannot assume that a CMV promoter that expresses well within the context of a viral vector, such as adenovirus, will function as well in a plasmid-based transfection system for the same cell context. Virus proteins produced by the viral vector are required for producing high levels of mRNA by the CMV promoter in specific cell nuclei.

Ideally, investigators design custom promoter enhancer chimeras that produce the highest levels of gene expression in their target cells of interest. Recently, we designed a systematic approach for customizing plasmids used for breast cancer gene therapy using expression profiling (18). Gene therapy clinical trials for cancer frequently produce inconsistent results. We believe that some of this variability could result from differences in transcriptional regulation that limit expression of therapeutic genes in specific cancers. Our systemic liposomal delivery of a nonviral plasmid DNA showed efficacy in animal models for several cancers. However, we observed large differences in the levels of gene expression from a CMV promoter enhancer between lung and breast cancers. To optimize gene expression in breast cancer cells in vitro and in vivo, we created a new promoter enhancer chimera to regulate gene expression. Serial analyses of gene expression data from a panel of breast carcinomas and normal breast cells predicted promoters that are highly active in breast cancers [e.g., the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter]. Furthermore, GAPDH is up-regulated by hypoxia, which is common in tumors. We added the GAPDH promoter, including the hypoxia enhancer sequences, to our in vivo gene expression plasmid. The novel CMV-GAPDH promoter enhancer showed up to 70-fold increased gene expression in breast tumors compared with the optimized CMV promoter enhancer alone. No significant increase in gene expression was observed in other tissues. These data demonstrate tissue-specific effects on gene expression after nonviral delivery and suggest that gene delivery systems may require plasmid modifications for the treatment of different tumor types. Furthermore, expression profiling can facilitate the design of optimal expression plasmids for use in specific cancers.

Several reviews have stated that nonviral systems are intrinsically inefficient compared with viral systems. However, as discussed above, one must separate issues of the delivery vehicle vs. the plasmid that is delivered. Case in point, we have shown that our extruded liposomes optimized for systemic delivery could out-compete delivery using a lentivirus. For example, we have compared SIVmac239, a highly noninfectious virus, with nonviral delivery of SIVmac239 DNA com-plexed to BIVs in adult rhesus macaques after injection into the saphenous vein of the leg. Our data showed that the monkeys injected with SIV DNA encapsulated in DOTAP:Chol BIVs were infected 4 days postinjection, and high levels of infection were produced in these monkeys at 14 days postinjection. Furthermore, higher levels of SIV RNA in the blood were produced using our BIV liposomes for delivery vs. using the SIV virus. CD4 counts were measured before and after injections. CD4 levels dropped in all monkeys to the lowest levels ever detected in the macaques in any experiment by 28 days postinjection, the first time point at which these counts were measured postinjection. All monkeys had clinical SIV infections and lost significant weight by day 28. These results were surprising because SIVmac239 is not highly infectious, and monkeys become sick with SIV infection only after several months or years postinjection with SIVmac239 virus. Therefore, we were able to induce SIV infection faster using our nonviral delivery of SIV plasmid DNA. In this case, we delivered a replication-competent plasmid so that gene expression increased over time posttransfection. Our delivery system was highly efficient and exceeded that of the lentivirus. The critical feature in this nonviral experiment was the plasmid DNA that was delivered.

Plasmids can be engineered to provide for specific or long-term gene expression, replication, or integration. Persistence elements, such as the inverted terminal repeats from adenovi-rus or adeno-associated virus, have been added to plasmids to prolong gene expression in vitro and in vivo. Apparently, these elements bind to the nuclear matrix, thereby retaining the plasmid in cell nuclei. For regulated gene expression, many different inducible promoters are used that promote expression only in the presence of a positive regulator or in the absence of a negative regulator. Tissue-specific promoters have been used for the production of gene expression exclusively in the target cells. As discussed in the previous paragraph, replication-competent plasmids or plasmids containing sequences for autonomous replication can be included that provide prolonged gene expression. Other plasmid-based strategies produce site-specific integration or homologous recombination within the host cell genome [reviewed in (42)]. Integration of a cDNA into a specific ''silent site'' in the genome could provide long-term gene expression without disruption of normal cellular functions. Homologous recombination could correct genetic mutations upon integration of wildtype sequences that replace mutations in the genome. Plasmids that contain fewer bacterial sequences and that produce high yield upon growth in Escherichia coli are also desirable.

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