Gene Transfer Process Research Articles

Nuclear entry of nonviral vectors

Nonviral gene delivery is limited to a large extent by multiple extracellular and intracellular barriers. One of the major barriers, especially in nondividing cells, is the nuclear envelope. Once in the cytoplasm, plasmids must make their way into the nucleus in order to be expressed. Numerous studies have demonstrated that transfections work best in dividing populations of cells in which the nuclear envelope disassembles during mitosis, thus largely eliminating the barrier. However, since many of the cells that are targets for gene therapy do not actively undergo cell division during the gene transfer process, the mechanisms of nuclear transport of plasmids in nondividing cells are of critical importance. In this review, we summarize recent studies designed to elucidate the mechanisms of plasmid nuclear import in nondividing cells and discuss approaches to either exploit or circumvent these processes to increase the efficiency of gene transfer and therapy.

Missing the Forest for the Trees: Phylogenetic Compression and Its Implications for Inferring Complex Evolutionary Histories

Phylogenetic tree reconstruction is difficult in the presence of lateral gene transfer and other processes generating conflicting signals. We develop a new approach to this problem using ideas borrowed from algorithmic information theory. It selects the hypothesis that simultaneously minimizes the descriptive complexity of the tree(s) plus the data when encoded using those tree(s). In practice this is the hypothesis that can compress the data the most. We show not only that phylogenetic compression is an efficient method for encoding most phylogenetic data sets and is more efficient than compression schemes designed for single sequences, but also that it provides a clear information theoretic rule for determining when a collection of conflicting trees is a better explanation of the data than a single tree. By casting the parsimony problem in this more general framework, we also conclude that the so-called total-evidence tree--the tree constructed from all the data simultaneously--is not always the most economical explanation of the data.

Bacterial death comes full circle: targeting plasmid replication in drug-resistant bacteria

It is now common for bacterial infections to resist the preferred antibiotic treatment. In particular, hospital-acquired infections that are refractory to multiple antibiotics and ultimately result in death of the patient are prevalent. Many of the bacteria causing these infections have become resistant to antibiotics through the process of lateral gene transfer, with the newly acquired genes encoding a variety of resistance-mediating proteins. These foreign genes often enter the bacteria on plasmids, which are small, circular, extrachromosomal pieces of DNA. This plasmid-encoded resistance has been observed for virtually all classes of antibiotics and in a wide variety of Gram-positive and Gram-negative organisms; many antibiotics are no longer effective due to such plasmid-encoded resistance. The systematic removal of these resistance-mediating plasmids from the bacteria would re-sensitize bacteria to standard antibiotics. As such, plasmids offer novel targets that have heretofore been unexploited clinically. This Perspective details the role of plasmids in multi-drug resistant bacteria, the mechanisms used by plasmids to control their replication, and the potential for small molecules to disrupt plasmid replication and re-sensitize bacteria to antibiotics. An emphasis is placed on plasmid replication that is mediated by small counter-transcript RNAs, and the "plasmid addiction" systems that employ toxins and antitoxins.

Gene Transfer with Three Amphiphilic Glycol Chitosans—the Degree of Polymerisation is the Main Controller of Transfection Efficiency

A number of studies have examined the possibility of delivering genes for the treatment of genetic diseases using various polymers and lipids. We have previously demonstrated the gene transfer ability of amphiphilic polymers (a soluble amine polymer covalently bound to lipid pendant groups). In the current communication we explore the gene transfer activity of amphiphilic glycol chitosans. Glycol chitosan was acid depolymerised to give polymers of various molecular weights. Palmitoyl or hexadecyl and in some cases additional N-methyl quaternary ammonium groups were attached to the polymers. DNA binding was studied by measuring the reduced fluorescence of ethidium bromide and the polyplex particle size and zeta potential. Biological characterisation of the polyplexes involved haemolysis, cytotoxicity and gene transfer assays. For the 22 polymers tested, DNA binding was optimum at a nitrogen to phosphate ratio of 2:1 and above. Polyplexes were 200–500 nm in diameter with a neutral or positive zeta potential. The haemolytic activity of the N-methyl polymers was studied and no haemolysis was detected up to a concentration of 10 mg ml−1. Cytotoxicity studies showed that the biocompatibility of glycol chitosan was adversely affected by a combination of a palmitoyl group and depolymerisation and that biocompatibility was subsequently restored with the introduction of N-methyl groups. In vitro transfection efficiency superior to the cationic lipid formulation N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulphate (DOTAP) was seen with depolymerised glycol chitosan in the A431 cell line only and with the depolymerised N-methyl quaternary ammonium amphiphilic derivatives in both the A431 and A549 cell lines. Degree of polymerisation (DP) was the most important controller of transfection efficiency and transfection resided within polymers with a DP of 73–171. High DP polymers diminished DNA–cell association, the first step in the cellular gene transfer process, thus apparently diminishing cell uptake. In vivo transfection with the N-methyl quaternary ammonium amphiphile was best at a DP of 86 and this glycol chitosan amphiphile gave superior liver and heart gene expression levels when compared to both Exgen 500 (linear polyethylenimine) and Superfect (a polyamidoamine dendrimer).

The evolution of molecular biology.

Biology's various affairs with holism and reductionism, and their contribution to understanding life at the molecular level

Mechanism of improved gene transfer by the N-terminal stearylation of octaarginine: enhanced cellular association by hydrophobic core formation.

The internalization mechanisms associated with octaarginine and stearyl-octaarginine were investigated using confocal laser microscopy and flow cytometric analysis. Octaarginine is able to translocate through cell membranes in a manner that does not exactly involve the classical endocytic pathways of internalization. However, when a stearyl moiety is attached to the N-terminus of octaarginine, the internalization shifts mainly to an endocytosis-dependent pathway. The transfection efficiency of stearyl-octaarginine was significantly higher than that of octaarginin. To understand the mechanism of the improved gene transfer by the N-terminal stearylation of octaarginine, the gene transfer processes mediated by octaarginine or stearyl-octaarginine were compared. Both octaarginine and stearyl-octaarginine are able to carry plasmid DNA into cells. The amount of plasmid DNA internalized as well as that delivered to the nucleus was higher in the case of stearyl-octaarginine. Even though the internalization mechanisms of octaarginine and stearyl-octaarginine were different, their complexes with plasmid DNA were internalized via the same pathway, presumably, the clathrin-mediated pathway of endocytosis. The results of the atomic force microscopy revealed that stearyl-octaarginine, but not octaarginine, can completely condense the DNA into stable complexes that can be highly adsorbed to the cell surface and subsequently highly internalized. Therefore, using stearylated-octaarginine provided higher internalization of plasmid DNA into cells, due to enhanced cellular association, as well as higher nuclear delivery. The results presented in this study provide a better understanding of the mechanisms of improved transfection using stearylated-octaarginine. The concept of using stearylated peptides may aid in the development of more efficient nonviral gene vectors.

Buffering properties of cationic polymethacrylates are not the only key to successful gene delivery.

Recently, we have shown that polymethacrylates containing imidazole side groups (HYMIMMA) or acid functions (MA), which have similar buffering properties as polyethyleneimine, were not able to transfect Cos-1 cells, whereas polymers containing only tertiary amines (DMAEMA) do transfect Cos-1 cells (Dubruel, P. et al. Eur. J. Pharm. Sci. 2003, 18 (3-4), 211-220). In the present work, we investigated to what extent the differences in transfection activity are related to differences in cellular internalization and/or subcellular localization. Therefore, we synthesized a series of polymethacrylates containing primary amine functions, used for the coupling of the fluorescent Oregon Green probe. The polymers containing acid functions were labeled with an amine containing fluorescein derivative (5-aminomethyl)fluorescein hydrochloride. It is demonstrated that the endosomal release of the MA and HYMIMMA-based complexes might be the limiting step in the gene transfer process in Cos-1 cells.

Investigating in situ natural genetic transformation of Acinetobacter sp. BD413 in biofilms with confocal laser scanning microscopy.

Natural genetic transformation is defined as the active uptake of free DNA, released in the environment through lysis or excretion, by bacterial strains that are naturally competent for transformation. It has been observed in a wide range of organisms (1). Of the three horizontal gene transfer processes — transformation, conjugation, and transduction — natural genetic transformation has the least requirements (1). For instance, there is no need for the donor cell to be alive or physically intact. Spatial and temporal separation between competent cells and the source of DNA in the environment can be overcome since nucleic acids are often found adsorbed to minerals (2), humic acids (3) or other components (1), where they are shielded from DNase attack. Consequently, DNA that survives can potentially be used for natural genetic transformation of recipient cells if those cells possess the ability to change into a state conferring competence for transformation (1). The process of transformation has been divided into the following steps (1,4,5): (i) release of DNA from cells; (ii) dispersal and (iii) persistence of the DNA in the environment; (iv) the development of competence for DNA uptake by cells in the natural habitat; (v) the interaction of cells with DNA and the uptake of DNA; and (vi) the expression of an acquired trait following DNA uptake (1).KeywordsConfocal Laser Scanning MicroscopyHorizontal Gene TransferTransformation FrequencyPeptide Nucleic AcidNucleic Acid StainThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Receptor-mediated gene transfer vectors: progress towards genetic pharmaceuticals.

Although specific delivery to tissues and unique cell types in vivo has been demonstrated for many non-viral vectors, current methods are still inadequate for human applications, mainly because of limitations on their efficiencies. All the steps required for an efficient receptor-mediated gene transfer process may in principle be exploited to enhance targeted gene delivery. These steps are: DNA/vector binding, internalization, subcellular trafficking, vesicular escape, nuclear import, and unpacking either for transcription or other functions (i.e., antisense, RNA interference, etc.). The large variety of vector designs that are currently available, usually aimed at improving the efficiency of these steps, has complicated the evaluation of data obtained from specific derivatives of such vectors. The importance of the structure of the final vector and the consequences of design decisions at specific steps on the overall efficiency of the vector will be discussed in detail. We emphasize in this review that stability in serum and thus, proper bioavailability of vectors to their specific receptors may be the single greatest limiting factor on the overall gene transfer efficiency in vivo. We discuss current approaches to overcome the intrinsic instability of synthetic vectors in the blood. In this regard, a summary of the structural features of the vectors obtained from current protocols will be presented and their functional characteristics evaluated. Dissecting information on molecular conjugates obtained by such methodologies, when carefully evaluated, should provide important guidelines for the creation of effective, targeted and safe DNA therapeutics.

RV002 Report on the activities of the national reference center for Borreliae

The Borrelia (B.) burgdorferi adhesin DbpA (decorin-binding protein A) is a valuable antigen for serodiagnosis of Lyme borreliosis and a promising candidate for a vaccine. To investigate the heterogeneity of DbpA, we aligned DNA sequences of 83 different dbpA genes (37 from the database, where the majority of sequences belong to B. burgdorferi sensu stricto and 46 were newly sequenced). Analysis of 25 sequences from the species B. burgdorferi s.s., 16 from B. afzelii, 40 from B. garinii, and two from the recently described human pathogenic genospecies A14S revealed five distinct DbpA groups. Group I comprises B. burgdorferi s.s. and group II B. afzelii. B. garinii is divided into groups III and IV, whereas A14S strains form group V. Formation of groups is mainly due to insertions of whole sequence sections. Comparison of dbpA sequences with ospC sequences from a subset of 59 strains revealed all kinds of cross-connections indicating processes of lateral gene transfer among strains. The extent of sequence identity within the dbpA genes decreases from the DNA (67%) to the amino acid (AA) level (44%) by about 23%, in contrast ospC sequence identities differed only by about 10%. This might be an indication that DbpA plays an important role in immune escape. Immunoblots using four recombinant DbpAs representing groups I–IV show that DbpA proteins are sensitive and specific antigens and complement one another in their reactivity. Part of the sera showed group-specific reactivity which could also be demonstrated with monoclonal antibodies.

Identification of human plasma proteins that bind to cationic lipid/DNA complex and analysis of their effects on transfection efficiency: implications for intravenous gene transfer

Interaction of cationic lipid/DNA complex with the plasma is a limiting step for the cationic lipid-mediated intravenous gene transfer and expression process. Most of the plasma components that interact with the complex and inhibit its transfection efficiency are still unknown. In the present work, human plasma proteins and lipoproteins that bind to a cationic lipid/DNA complex were isolated on a sucrose density gradient and identified by 2-D gel electrophoresis. Protein binding did not result in complex dissociation or DNA degradation. The effects of several complex-binding plasma components on the transfection efficiency were studied using lung endothelial cells cultured in vitro. Lipoprotein particles caused a drastic loss of the transfection efficiency of the complex. Surprisingly, fibrinogen was found to activate the transfection process. The roles of these complex-binding plasma components on the complex uptake efficiency were quantitatively assessed using radiolabeled plasmid DNA and qualitatively evaluated using fluorescence microscopy. A good correlation was found between the effects of the complex-binding plasma components on the transfection and on cell uptake efficiencies. In contrast to what was generally believed, our data suggest that disruption of the complex does not occur when it is in contact with the plasma and therefore could not be responsible for the loss of transfection activity. Instead, coating of complexes with plasma components seems to be responsible for reduced uptake by cells, which in turn results in reduced transfection.

The effects of cyclic stretch on gene transfer in alveolar epithelial cells

Cyclic stretch has been shown to alter cell physiology, cytoskeletal structure, signal transduction, and gene expression in a variety of cell types. To determine the effects of stretch on the gene transfer process, we compared the transfection efficiencies of human A549 cells grown either statically or exposed to 10% cyclic stretch (Δ surface area) at 60 cycles/min (1 Hz) for 24 hours prior to, and/or after transfection with pEGFP-N1 and pCMV-lux-DTS using lipoplex or electroporation. Stretching the cells prior to transfection had no effect on gene transfer. By contrast, cyclic, but not continuous, stretch applied immediately after transfection for as little as 30 minutes resulted in a 10-fold increase in gene transfer and expression by either transfection technique. These stretch conditions did not result in rupture of the plasma membrane based on the fact that DNA was unable to enter stretched cells unless either an electric field was applied or the DNA was complexed with liposomes. Taken together with the timing of the stretch response and the known effects of stretch on transcription, these findings suggest that cyclic stretch may be altering the intracellular transport of plasmids to increase gene expression.

Horizontal gene transfer in the phytosphere

Here, the ecological aspects of gene transfer processes between bacteria in the phytosphere are examined in the context of emerging evidence for the dominant role that horizontal gene transfer (HGT) has played in the evolutionary shaping of bacterial communities. Moreover, the impact of the putative capture of genetic material by bacteria from plants is discussed. Examples are provided that illustrate how mobile genetic elements (MGEs) influence the behaviour of bacteria in their natural habitat, especially in structured communities such as biofilms on plant surfaces. This community behaviour is used as a framework to pose questions on the evolutionary role and significance of gene transfer processes in plant-associated habitats. Selection within the highly structured phytosphere is likely to represent a dominant force shaping the genetic make-up of plant-associated bacterial communities. Current understanding of the triggering and impact of horizontal gene transfer, however, remains limited by our lack of understanding of the nature of the selective forces that act on bacteria in situ. The individual, colony, population and community level selection benefits imposed by the ability to use specific carbon sources or survive selective compounds are clear, but it is not always possible to assess what drives gene transfer and persistence. The role of HGT in the adaptation of host bacteria to their environmental niche is still not fully understood.

Calorimetry of Cationic Liposome–DNA Complex and Intracellular Visualization of the Complexes

This chapter describes cationic liposome–mediated gene transfer that has become a widely used tool for in vitro transfection of eukaryotic cells and as a promising and safe alternative for in vivo gene therapy applications. It involves the formation of a cationic lipid–DNA complex that interacts efficiently with the cell surface, leading to the entry and expression of the exogenous DNA. The use of cationic lipids as DNA delivery systems for gene therapy is limited by the relatively low efficiency of the gene transfer process compared with viral vectors. Improvement of the in vitro and in vivo cationic lipid–mediated gene transfection efficiency can be attempted by use of trial and error approaches consisting of synthesizing and testing a large number of cationic lipid derivatives. Alternatively, a deeper understanding of the intermediate steps and mechanisms involved in the gene transfer process mediated by cationic lipids could allow the design of new rational strategies to improve the transfection efficiency. Two key steps are involved in the cationic lipid–mediated gene transfer process: (1) interaction between the cationic liposomes and DNA leading to the formation of a complex and (2) interaction between the complex and the target cells leading to DNA entry and expression of the transgene.

The ecology of transfer of mobile genetic elements

The ecological aspects of the transfer and spread of mobile genetic elements (MGE) are reviewed in the context of the emerging evidence for the dominant role that horizontal gene transfer (HGT) has played in the evolutionary shaping of bacterial communities. Novel tools are described that allow a refined analysis of HGT in natural settings. The occurrence of HGT processes in soil and water, as affected by environmental factors, is then discussed. Examples are provided that illustrate how MGE can influence the behavior of microorganisms in their natural habitats. The occurrence of microorganisms as groups of cells in structured communities, such as those found in biofilms, is used as a framework in order to review the data and pose further questions on the evolutionary role and significance of contemporary gene transfer processes in nature. Selection by the environment is likely to be the dominant force in shaping the genetic make-up of bacterial communities. In fact, selective force can act as an apparent accelerator of gene transfer processes, mainly as a result of the enhancement of survival and persistence of favorably selected products of gene transfer processes (genes, metabolic pathways, microbial cells and communities). However, the current understanding of the triggering and impact of HGT in nature remains limited by our lack of understanding of the very nature and variety of the selective forces that act on microorganisms in situ. Hence, the relevant questions with respect to these triggers acting in natural habitats need to be answered using advanced approaches for studying HGT processes in nature, such as those discussed in this review.

An experimental approach for direct observation of the interaction of polyanions with sphingosine-containing giant vesicles

A new approach for direct optical microscopy observation of polyanion interactions with bilayers of giant cationic liposomes (GUVs) was suggested. Polyanions as DNA, dextran sulfate (DS), heparin (H) and polyacrylic acids (PA) were locally delivered by a micropipette to a part of a giant unilamellar vesicle membrane. The phenomena were directly observed under optical microscope. GUVs, about 100 μm in diameter, formed of phosphatidylcholines and up to 33 mol% of the natural bioactive cationic amphiphile sphingosine (Sph), were prepared by electroformation. The effects of water-soluble molecules with high negative linear charge density as dextran sulfate (DS), heparin (H) polyacrylic acids (PA) and adenosine-5′-triphosphoric acid (ATP) were compared with those of DNAs. The resulting membrane topology transformations were monitored in phase contrast, while the DNA distribution was followed in fluorescence. DNA-induced endocytosis-like membrane morphology transformation due to the DNA/lipid membrane local interactions was observed. The DS, H and PA induced membrane topology transformations similar to those of the DNAs, while ATP did not cause any detectable ones. The endocytosis mechanism involves the formation of ordered domains in the GUV membrane where some surface and charge asymmetries between the two membrane monolayers were created. The sizes of created polyanionic/cationic membrane domains depend on the form, length and elasticity of the adsorbed highly charged molecules. Endosome-including capacities of polyanionic molecules depend heavily on the high linear negative charge at a certain length. An original method for direct studying of the DNA/membrane interactions in autoadaptable giant liposome system imitating biological membrane interactions was forwarded. The model observations could also help for understanding events associated with cationic liposome/DNA complex formation in gene transfer processes.

Microseeding: Gene Transfer for Modulation of Wound Healing

Gene transfer has many areas of potential use in tissue repair such as skin including wounded skin, muscle, tendon, and bone. Particularly in vivo techniques, but also combinations of in vivo and in vitro techniques, are of interest to plastic surgeons. For in vivo gene transfer to skin and wound, single subepidermal injection, the gene gun technique, or microseeding can be used. Single injection and microseeding have the advantage that not only plasma DNA, but also liposomal and viral constructs can be used. The yield is quite high, particularly when, for instance, microseeding is combined with an adenoviral vector. Gene transfer to partial thickness wounds has been done with the gene gun technique resulting in a significant acceleration of healing. For full thickness wounds, microseeding will yield high expression of protein in the wound environment. Our group has been using a chamber technique in order to contain the gene transfer process, the expressed protein, and the wound itself. Gene transfer to bone is major interest, particularly for delivery of proteins that can upregulate the repair process in bone. Most delivery techniques call for an in vitro gene transfer protocol, usually targeting harvested bone marrow cells and these cells are autotransplanted during surgical exposure of the area that will undergo repair. Using microseeding technique, it is possible to transfect the inside of the periosteum and in this fashion, deliver proteins in high local concentrations. The technique has been used successfully in experimental research targeting the periosteum in the tibial and femoral periosteum, as well as the dento-alveolar membrane with high expression. Our group has also worked with a tetracycline repressor system for control of timing and amount of gene expression in various cells. This system is useful, both when it is desired that the expression of the delivered gene is delayed and also allowing for more precise control of the expression of a particular gene.

Evolution of the enzymes of the citric acid cycle and the glyoxylate cycle of higher plants. A case study of endosymbiotic gene transfer.

The citric acid or tricarboxylic acid cycle is a central element of higher-plant carbon metabolism which provides, among other things, electrons for oxidative phosphorylation in the inner mitochondrial membrane, intermediates for amino-acid biosynthesis, and oxaloacetate for gluconeogenesis from succinate derived from fatty acids via the glyoxylate cycle in glyoxysomes. The tricarboxylic acid cycle is a typical mitochondrial pathway and is widespread among alpha-proteobacteria, the group of eubacteria as defined under rRNA systematics from which mitochondria arose. Most of the enzymes of the tricarboxylic acid cycle are encoded in the nucleus in higher eukaryotes, and several have been previously shown to branch with their homologues from alpha-proteobacteria, indicating that the eukaryotic nuclear genes were acquired from the mitochondrial genome during the course of evolution. Here, we investigate the individual evolutionary histories of all of the enzymes of the tricarboxylic acid cycle and the glyoxylate cycle using protein maximum likelihood phylogenies, focusing on the evolutionary origin of the nuclear-encoded proteins in higher plants. The results indicate that about half of the proteins involved in this eukaryotic pathway are most similar to their alpha-proteobacterial homologues, whereas the remainder are most similar to eubacterial, but not specifically alpha-proteobacterial, homologues. A consideration of (a) the process of lateral gene transfer among free-living prokaryotes and (b) the mechanistics of endosymbiotic (symbiont-to-host) gene transfer reveals that it is unrealistic to expect all nuclear genes that were acquired from the alpha-proteobacterial ancestor of mitochondria to branch specifically with their homologues encoded in the genomes of contemporary alpha-proteobacteria. Rather, even if molecular phylogenetics were to work perfectly (which it does not), then some nuclear-encoded proteins that were acquired from the alpha-proteobacterial ancestor of mitochondria should, in phylogenetic trees, branch with homologues that are no longer found in most alpha-proteobacterial genomes, and some should reside on long branches that reveal affinity to eubacterial rather than archaebacterial homologues, but no particular affinity for any specific eubacterial donor.

Role of intracellular cationic liposome-DNA complex dissociation in transfection mediated by cationic lipids.

The cationic lipid-mediated gene transfer process involves sequential steps: internalization of the cationic lipid-DNA complexes inside the cells via an endocytosis-like mechanism, escape from endosomes, dissociation of the complex, and finally entry of free DNA into the nucleus. However, cationic lipid-DNA complex dissociation in the cytoplasm and the ability of the subsequently released DNA to enter the nucleus have not yet been demonstrated. In this report we showed, using confocal laser scanning analysis, that microinjection of a double fluorescent-labeled cationic lipid-pCMV-LacZ plasmid complex into the cytoplasm of HeLa cells results in efficient complex dissociation. However, the released DNA did not enter the nucleus, and no significant transfection could be detected. In contrast, nuclear microinjection of the cationic lipid-pCMV-LacZ plasmid complex resulted in efficient complex dissociation and transfection of all the cells. Taken together, the data suggest that intracellular dissociation of the cationic lipid-DNA complex is not a limiting step for transfection as previously thought.

Helper virus-free HSV-1 vectors packaged both in the presence of VSV G protein and in the absence of HSV-1 glycoprotein B support gene transfer into neurons in the rat striatum.

Herpes simplex virus (HSV-1) vectors have potential for gene transfer into quiescent cells, but the gene transfer process could be more efficient. In other vector systems, both the titers and the efficiency of gene transfer have been enhanced by pseudotyping the vector particles with vesicular stomatitis virus (VSV) G protein. In this report, we pseudotyped helper virus-free HSV-1 plasmid vectors with VSV G protein. Packaging was performed in the presence of both VSV G protein and a deletion in an essential HSV-1 glycoprotein, gB. The resulting vector stocks supported gene transfer into both fibroblast and neuronal cell lines. VSV G protein was required for gene transfer because preincubation of these vector stocks with antibodies directed against either VSV G protein or VSV reduced the titer to undetectable levels. Although the titers were lower than those obtained using the unmodified vector system, the titers were not increased by use of chimeric proteins that contain the extracellular domain of VSV G protein and the transmembrane and/or cytoplasmic domains of specific HSV-1 glycoproteins. Also, the titers were not increased by performing the packaging in the presence of deletions in multiple HSV-1 glycoproteins. Nonetheless, pHSVlac pseudotyped with VSV G protein supported gene transfer into striatal neurons in the rat brain. Thus, HSV-1 vectors pseudotyped with VSV G protein may be useful for specific gene transfer studies.