"Alternative" endocytic mechanisms exploited by pathogens: new avenues for therapeutic delivery?

Some pathogens utilize unique routes to enter cells that may evade the intracellular barriers encountered by the typical clathrin-mediated endocytic pathway. Retrograde transport and caveolar uptake are among the better characterized pathways, as alternatives to clathrin-mediated endocytosis, that are known to facilitate entry of pathogens and potential delivery agents. Recent characterization of the trafficking mechanisms of prion proteins and certain bacteria may present new paradigms for strategizing improvements in therapeutic spread and retention of therapy. This review will provide an overview of such endocytic pathways, and discuss current and future possibilities in using these routes as a means to improve therapeutic delivery.

Typical and atypical trafficking pathways of Ad5 penton base recombinant protein: implications for gene transfer.

The adenovirus (Ad) penton base protein facilitates viral infection by binding cell surface integrins, triggering receptor-mediated endocytosis and mediating endosomal penetration. Given these multiple functions, recombinant penton base proteins have been utilized as non-viral vehicles for gene transfer by our lab and others. Although we have previously demonstrated that penton base-derived vectors undergo integrin-specific binding and cell entry, less than desirable levels of gene expression have led us to re-evaluate the recombinant penton base as an agent for gene delivery. To do so, we have examined here the intracellular trafficking of an Ad serotype 5 (Ad5) recombinant penton base protein (PB). Here, we not only observed that PB utilizes a similar, typical trafficking pathway of whole Ad, but also found that PB entered HeLa cells through pathways not yet identified as contributing to cell entry by the whole virus. We show by high-resolution confocal microscopy and biochemical methods that binding to alphav-integrins is a requirement for cell entry, but that early internalization stages did not substantially pass through clathrin-positive and early endosomal compartments. Moreover, a subpopulation of internalized protein localized with caveolin-positive compartments and Golgi markers, suggesting that a certain percentage of proteins pass through non-clathrin-mediated pathways. Similar to the virus, trafficking toward the nucleus was affected by disruption of microtubules and dynein. The majority of penton base molecules avoided the lysosome while facilitating early vesicle release of low molecular weight dextran molecules. In further support of a vesicle escape capacity, a subpopulation of internalized penton base appeared to enter the nucleus, as observed by high-resolution confocal microscopy and cell fractionation. As a confirmation of these findings, we demonstrate that a recombinant penton base facilitated cytosolic entry of an siRNA molecule as observed by RNA interference of a marker gene. Based on our findings here, we suggest that whereas soluble penton base proteins may enter cells through clathrin- and non-clathrin-mediated pathways, vesicle escape and nuclear delivery appear to be supported by a clathrin-mediated pathway. As our previous efforts have focused on utilizing recombinant penton base proteins as delivery agents for therapeutics, these findings allow us to evaluate the use of the penton base as a cell entry and intracellular trafficking agent, and may be of interest concerning the development of vectors for efficient delivery of therapeutics to cells.

Intracellular trafficking of nonviral vectors.

Nonviral vectors continue to be attractive alternatives to viruses due to their low toxicity and immunogenicity, lack of pathogenicity, and ease of pharmacologic production. However, nonviral vectors also continue to suffer from relatively low levels of gene transfer compared to viruses, thus the drive to improve these vectors continues. Many studies on vector-cell interactions have reported that nonviral vectors bind and enter cells efficiently, but yield low gene expression, thus directing our attention to the intracellular trafficking of these vectors to understand where the obstacles occur. Here, we will review nonviral vector trafficking pathways, which will be considered here as the steps from cell binding to nuclear delivery. Studies on the intracellular trafficking of nonviral vectors has given us valuable insights into the barriers these vectors must overcome to mediate efficient gene transfer. Importantly, we will highlight the different approaches used by researchers to overcome certain trafficking barriers to gene transfer, many of which incorporate components from biological systems that have naturally evolved the capacity to overcome such obstacles. The tools used to study trafficking pathways will also be discussed.

The Ad5 fiber mediates nonviral gene transfer in the absence of the whole virus, utilizing a novel cell entry pathway.

The interesting discovery reported here that soluble adenovirus serotype 5 (Ad5) fiber proteins enter cells without the virus was a serendipitous result during our development of Ad5 capsid proteins as nonviral gene transfer vectors. The Ad5 capsid fiber and penton proteins mediate infection. The fiber docks to a noninternalizing cell surface protein called the coxsackievirus-Ad receptor (CAR), followed by penton binding to integrins, triggering integrin-mediated endocytosis of the virus. In our previous work, we assembled the nonviral complex, 3PO, which utilized the penton to mediate gene transfer through integrin binding and endocytosis. Here, we tested whether incorporating the fiber targets 3PO to CAR, thus recapitulating the Ad5 infection pathway. As CAR is not an endocytic receptor, we were surprised to find that the fiber alone, without the penton, enabled gene transfer by binding CAR, but internalizing through an unknown mechanism. We show here that the fiber distributes to the nucleus and cytoplasm after temperature-independent uptake, whereas the penton accumulates around the nucleus after temperature-dependent uptake. Fiber uptake by HeLa cells is also actin-dependent, requires the fiber tail/shaft region, and is largely inhibited by heparin. This study raises the possibility that alternative pathways may enable both viral and nonviral cell entry.

Adenoviral capsid modulates secretory compartment organization and function in acinar epithelial cells from rabbit lacrimal gland.

Although adenovirus (Ad) exhibits tropism for epithelial cells, little is known about the cellular effects of adenoviral binding and internalization on epithelial functions. Here, we examine its effects on the secretory acinar epithelial cells of the lacrimal gland, responsible for stimulated release of tear proteins into ocular fluid. Exposure of reconstituted rabbit lacrimal acini to replication-defective Ad for 16-18 h under conditions that resulted in >80% transduction efficiency did not alter cytoskeletal filament or biosynthetic/endosomal membrane compartment organization. Transduction specifically altered the organization of the stimulated secretory pathway, eliciting major dispersal of rab3D immunofluorescence from apical stores normally associated with mature secretory vesicles. Biochemical studies revealed that this dispersal was not associated with altered rab3D expression nor its release from cellular membranes. Ultraviolet (UV)-inactivated Ad elicited similar dispersal of rab3D immunofluorescence. In acini exposed to replication-defective or UV-inactivated Ad, carbachol-stimulated release of bulk protein and beta-hexosaminidase were significantly (P< or =0.05) inhibited to an extent proportional to the loss of rab3D-enriched mature secretory vesicles associated with these treatments. We propose that the altered secretory compartment organization and function caused by Ad reflects changes in the normal maturation of secretory vesicles, and that these changes are caused by exposure to the Ad capsid.

3PO, a novel nonviral gene delivery system using engineered Ad5 penton proteins.

This study describes the development of 3PO, a nonviral, protein-based gene delivery vector which utilizes the highly evolved cell-binding, cell-entry and intracellular transport functions of the adenovirus serotype 5 (Ad5) capsid penton protein. A penton fusion protein containing a polylysine sequence was produced by recombinant methods and tested for gene delivery capability. As the protein itself is known to bind integrins through a conserved consensus motif, the penton inherently possesses the ability to bind and enter cells through receptor-mediated internalization. The ability to lyse the cellular endosome encapsulating internalized receptors is also attributed to the penton. The recombinant protein gains the additional function of DNA binding and transport with the appendage of a polylysine motif. This protein retains the ability to form pentamers and mediates delivery of a reporter gene to cultured cells. Interference by oligopeptides bearing the integrin binding motif suggests that delivery is mediated specifically through integrin receptor binding and internalization. The addition of protamine to penton-DNA complexes allows gene delivery in the presence of serum.

Nonviral gene delivery to human breast cancer cells by targeted Ad5 penton proteins.

The capsid proteins of adenovirus serotype 5 (Ad5) are key to the virus' highly efficient cell binding and entry mechanism. In particular, the penton base plays a significant role in both viral internalization and endosome penetration. We have produced an adenovirus penton fusion protein (HerPBK10) containing moieties for DNA transport and targeted delivery to breast cancer cells. HerPBK10 binds DNA through a polylysine appendage, while the EGF-like domain of the heregulin-alpha(1) isoform is used as the targeting ligand. This ligand binds with high affinity to HER2/3 or HER2/4 heterodimers, which are overexpressed on certain aggressive breast cancers. In addition, this ligand is rapidly internalized after binding, thus adding to the utility of heregulin for targeting. HerPBK10 binds MDA-MB-453 breast cancer cells in a receptor-specific manner, and mediates the entry of a reporter plasmid in MDA-MB-453 cells in culture. Delivery can be competed by excess heregulin peptide, thus confirming receptor specificity. Importantly, the penton segment appears to contribute significantly to enhanced delivery. Complexes containing HerPBK10 and DNA have been optimized to provide targeted gene delivery to breast cancer cells in vitro. We demonstrate that delivery can be accomplished in the presence of serum, thus suggesting a potential use for in vivo delivery.

Assessing the binding and endocytosis activity of cellular receptors using GFP-ligand fusions.

We have developed a simple scheme for characterizing ligand-receptor binding and post-binding activity on living cells. Our approach makes use of green fluorescent protein (GFP) as an auto-fluorescent tag to label protein ligands. We have constructed GFP-tagged ligands that can be expressed in bacteria as soluble fusion proteins. A cell-binding assay using fluorescence-activated cell sorting (FACS) demonstrates that GFP-tagged proteins retain their wild-type receptor-binding specificity. Using this assay, we measure ligand binding on unfixed cells and demonstrate receptor specificity using specific competitors. To determine the ability of receptor targets to internalize, we developed a second FACS-based assay to detect the rate and percentage of internalized ligand in living cells. Noninternalizing control ligands and fluorescence microscopy of treated cells confirm that our assay is reliable for determining receptor internalization activity.

4-Aminobutyrate aminotransferase (GABA-transaminase) deficiency.

4-Aminobutyrate aminotransferase (GABA-transaminase, GABA-T, EC 2.6.1.19) deficiency (McKusick 137150), an inborn error of GABA degradation, has until now been documented in only a single Flemish child. Compared to the other defects of GABA degradation, succinic semialdehyde dehydrogenase (SSADH, EC 1.2.1.24) deficiency with > 150 patients (McKusick 271980) and pyridoxine-dependent seizures with > 100 patients ('putative' glutamic acid decarboxylase (GAD, EC 4.1.1.15) deficiency; McKusick 266100), GABA-T deficiency is very rare. We present a summary of the clinical, biochemical, enzymatic and molecular findings on the index proband, and a recently identified second patient, with GABA-T deficiency. The phenotype in both included psychomotor retardation, hypotonia, hyperreflexia, lethargy, refractory seizures and electroencephalographic abnormalities. In an effort to elucidate the molecular basis of GABA-T deficiency, we isolated and characterized a 1.5 kb cDNA encoding human GABA-T, in addition to a 41 kb genomic clone which encompassed the GABA-T coding region. Standard methods of cloning and sequencing revealed an A-to-G transition at nucleotide 754 of the coding region in lymphoblast cDNAs derived from the index proband. This mutation resulted in substitution of an invariant arginine at amino acid 220 by lysine. Expression of the mutant in E. coli, followed by isolation and enzymatic characterization of the recombinant protein, revealed an enzyme whose Vmax was reduced to 25% of wild-type activity. The patient and father were heterozygous for this allele; the second allele in the patient remains unidentified. Genomic Southern analysis revealed that the second proband most likely harbours a deletion in the 3' region of the GABA-T gene.

Identification of a familial mutation associated with GABA-transaminase deficiency disease.

GABA-transaminase (GABA-T) deficiency disease is a rare recessive disorder characterized by abnormal development, seizures, and high levels of GABA in serum and cerebrospinal fluid. Although some patients are the offspring of consanguineous marriages, most are not. To identify the molecular basis of this disease, we have determined the sequence of human GABA-T cDNA. We have compared the GABA-T cDNA sequences in cultured cells derived from six healthy controls with those from a GABA-T-deficient patient and both parents. Our data indicate that GABA-T deficiency disease may result from an allele that encodes an R220K substitution.

A rat brain cDNA encodes enzymatically active GABA transaminase and provides a molecular probe for GABA-catabolizing cells.

cDNAs encoding gamma-aminobutyric acid aminotransferase (GABA-T) were isolated from a lambda ZAP rat hippocampal cDNA expression library by two independent cloning methods, immunological screening with an antimouse GABA-T antibody and plaque hybridization with a GABA-T cDNA probe derived by polymerase chain reaction. We have produced enzymatically active GABA-T from a rat brain cDNA containing the full-length GABA-T coding region. Our rat brain GABA-T cDNAs hybridize to mRNAs in brain and peripheral tissues, including liver, kidney, and testis. We have also detected GABA-T mRNA in GABAergic cells of rat cerebellar cortex by in situ hybridization. Our rat brain GABA-T probe hybridizes to Purkinje, basket, stellate, and Golgi II cells, the same GABAergic neurons previously shown to contain glutamate decarboxylase GAD65 and GAD67.