Charge-dependent translocation of Bordetella pertussis adenylate cyclase toxin into eukaryotic cells: Implication for the in vivo delivery of CD8+ T cell epitopes into antigen-presenting cells

Bordetella pertussis secretes a calmodulin-activated adenylate cyclase toxin, CyaA, that is able to deliver its N-terminal catalytic domain (400-aa residues) into the cytosol of eukaryotic target cells, directly through the cytoplasmic membrane. We have previously shown that CyaA can be used as a vehicle to deliver T cell epitopes, inserted within the catalytic domain of the toxin, into antigen-presenting cells and can trigger specific class I-restricted CD8+ cytotoxic T cell responses in vivo. Here, we constructed a series of recombinant toxins harboring at the same insertion site various peptide sequences of 11–25 amino acids, corresponding to defined CD8+ T cell epitopes and differing in the charge of the inserted sequence. We show that inserted peptide sequences containing net negative charges (−1 or −2) decreased or completely blocked (charge of −4) the internalization of the toxin into target cells in vitro and abolished the induction of cytotoxic T cell responses in vivo. The blocking of translocation due to the inserted acidic sequences can be relieved by appropriate mutations in the flanking region of CyaA that counterbalance the inserted charges. Our data indicate that (i) the electrostatic charge of the peptides inserted within the catalytic domain of CyaA is critical for its translocation into eukaryotic cells and (ii) the delivery of T cell epitopes into the cytosol of antigen-presenting cells by recombinant CyaA toxins is essential for the in vivo stimulation of specific cytotoxic T cells. These findings will help to engineer improved recombinant CyaA vectors able to stimulate more efficiently cellular immunity.

Administration of helper-dependent adenoviral vectors and sequential delivery of different vector serotype for long-term liver-directed gene transfer in baboons

The efficiency of first-generation adenoviral vectors as gene delivery tools is often limited by the short duration of transgene expression, which can be related to immune responses and to toxic effects of viral proteins. In addition, readministration is usually ineffective unless the animals are immunocompromised or a different adenovirus serotype is used. Recently, adenoviral vectors devoid of all viral coding sequences (helper-dependent or gutless vectors) have been developed to avoid expression of viral proteins. In mice, liver-directed gene transfer with AdSTK109, a helper-dependent adenoviral (Ad) vector containing the human α1-antitrypsin (hAAT) gene, resulted in sustained expression for longer than 10 months with negligible toxicity to the liver. In the present report, we have examined the duration of expression of AdSTK109 in the liver of baboons and compared it to first-generation vectors expressing hAAT. Transgene expression was limited to approximately 3–5 months with the first-generation vectors. In contrast, administration of AdSTK109 resulted in transgene expression for longer than a year in two of three baboons. We have also investigated the feasibility of circumventing the humoral response to the virus by sequential administration of vectors of different serotypes. We found that the ineffectiveness of readministration due to the humoral response to an Ad5 first-generation vector was overcome by use of an Ad2-based vector expressing hAAT. These data suggest that long-term expression of transgenes should be possible by combining the reduced immunogenicity and toxicity of helper-dependent vectors with sequential delivery of vectors of different serotypes.

Short-term correction of factor VIII deficiency in a murine model of hemophilia A after delivery of adenovirus murine factor VIII in utero

Development of in utero gene transfer approaches may provide therapies for genetic disorders with perinatal morbidity. In hemophilia A, prenatal and postnatal bleeding may be catastrophic, and modest increments in factor VIII (FVIII) activity are therapeutic. We performed transuterine i.p. gene transfer at day 15 of gestation in a murine model of hemophilia A. Normal, carrier (XHX), and FVIII-deficient (XHY and XHXH) fetuses injected with adenoviral vectors carrying luciferase or β-galactosidase reporter genes showed high-level gene expression with 91% fetal survival. The live-born rates of normal and FVIII-deficient animals injected in utero with adenovirus murine FVIII (3.3 × 105 plaque-forming units) was 87%. FVIII activity in plasma was 50.7 ± 10.5% of normal levels at day 2 of life, 7.2 ± 2.2% by day 15 of life, and no longer detectable at day 21 of life in hemophilic animals. Injection of higher doses of murine FVIII adenovirus at embryonic day 15 produced supranormal levels of FVIII activity in the neonatal period. PCR analysis identified viral genomes primarily in the liver, intestine, and spleen, although adenoviral DNA was detected in distal tissues when higher doses of adenovirus were administered. These studies show that transuterine i.p. injection of adenoviral vectors produces therapeutic levels of circulating FVIII throughout the neonatal period. The future development of efficient and persisting vectors that produce long-term gene expression may allow for in utero correction of genetic diseases originating in the fetal liver, hematopoietic stem cells, as well as other tissues.

In vivo NMR and MRI using injection delivery of laser-polarized xenon

Because xenon NMR is highly sensitive to the local environment, laser-polarized xenon could be a unique probe of living tissues. Realization of clinical and medical science applications beyond lung airspace imaging requires methods of efficient delivery of laser-polarized xenon to tissues, because of the short spin-lattice relaxation times and relatively low concentrations of xenon attainable in the body. Preliminary results from the application of a polarized xenon injection technique for in vivo 129Xe NMR/MRI are extrapolated along with a simple model of xenon transit to show that the peak local concentration of polarized xenon delivered to tissues by injection may exceed that delivered by respiration by severalfold.

Delivery of a stringent dimerizer-regulated gene expression system in a single retroviral vector

Small molecule-regulated transcription has broad utility and would benefit from an easily delivered self-contained regulatory cassette capable of robust, tightly controlled target gene expression. We describe the delivery of a modified dimerizer-regulated gene expression system to cells on a single retrovirus. A transcription factor cassette responsive to the natural product dimerizer rapamycin was optimized for retroviral delivery by fusing a highly potent chimeric activation domain to the rapamycin-binding domain of FKBP-rapamycin-associated protein (FRAP). This improvement led to an increase in both the potency and maximal levels of gene expression induced by rapamycin, or nonimmunosuppressive rapamycin analogs. The modified transcription factor cassette was incorporated along with a target gene into a single rapamycin-responsive retrovirus. Cell pools stably transduced with the single virus system displayed negligible basal expression and gave induction ratios of at least three orders of magnitude in the presence of rapamycin or a nonimmunosuppressive rapamycin analog. Levels of induced gene expression were comparable to those obtained with the constitutive retroviral long terminal repeat and the single virus system performed well in four different mammalian cell lines. Regulation with the dimerizer-responsive retrovirus was tight enough to allow the generation of cell lines displaying inducible expression of the highly toxic diphtheria toxin A chain gene. The ability to deliver the tightly inducible rapamycin system in a single retrovirus should facilitate its use in the study of gene function in a broad range of cell types.

Hydrolysis of GTP on rab11 is required for the direct delivery of transferrin from the pericentriolar recycling compartment to the cell surface but not from sorting endosomes

Rab11 is a small GTP-binding protein that in cultured mammalian cells has been shown to be concentrated in the pericentriolar endosomal recycling compartment and to play a key role in passage of the recycling transferrin receptor through that compartment [Ullrich, O., Reinsch, S., Urbé, S., Zerial, M. & Parton, R. G. (1996) J. Cell Biol. 135, 913–924]. To obtain insights into the site(s) of action of rab11 within the recycling pathway, we have now compared the effects on recycling at 37°C of overexpression of wild-type rab11 and various mutant forms of this protein in cells that had been loaded with transferrin at either 37°C or 16°C. We show that incubation at 16°C blocks passage of endocytosed transferrin into the recycling compartment and that, whereas the rab11 dominant negative mutant form (S25N) inhibits transferrin recycling after interiorization at either temperature, the wild-type rab11 and constitutively active mutant (Q70L) have no inhibitory effect on the recycling of molecules that were interiorized at 16°C. This differential inhibitory effect shows that two distinct pathways for recycling are followed by the bulk of the transferrin molecules interiorized at the two different temperatures. The incapacity of the constitutively active form of rab11 (Q70L) to inhibit recycling of molecules interiorized at 16°C is consistent with their recycling taking place directly from sorting endosomes, in a process that does not require hydrolysis of GTP on rab11. The fact that the dominant negative (S25N) form of rab11 inhibits recycling of molecules interiorized at both temperatures indicates that activation of rab11 by GTP is required for exit of transferrin from sorting endosomes, regardless of whether this exit is toward the recycling compartment or directly to the plasma membrane.

Scavenger receptor class B, type I (SR-BI) is the major route for the delivery of high density lipoprotein cholesterol to the steroidogenic pathway in cultured mouse adrenocortical cells

The class B, type I scavenger receptor, SR-BI, binds high density lipoprotein (HDL) and mediates the selective uptake of HDL cholesteryl ester (CE) by cultured transfected cells. The high levels of SR-BI expression in steroidogenic cells in vivo and its regulation by tropic hormones provides support for the hypothesis that SR-BI is a physiologically relevant HDL receptor that supplies substrate cholesterol for steroid hormone synthesis. This hypothesis was tested by determining the ability of antibody directed against murine (m) SR-BI to inhibit the selective uptake of HDL CE in Y1-BS1 adrenocortical cells. Anti-mSR-BI IgG inhibited HDL CE-selective uptake by 70% and cell association of HDL particles by 50% in a dose-dependent manner. The secretion of [3H]steroids derived from HDL containing [3H]CE was inhibited by 78% by anti-mSR-BI IgG. These results establish mSR-BI as the major route for the selective uptake of HDL CE and the delivery of HDL cholesterol to the steroidogenic pathway in cultured mouse adrenal cells.

Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O

The pore-forming toxin streptolysin O (SLO) can be used to reversibly permeabilize adherent and nonadherent cells, allowing delivery of molecules with up to 100 kDa mass to the cytosol. Using FITC-labeled albumin, 105–106 molecules were estimated to be entrapped per cell. Repair of toxin lesions depended on Ca2+-calmodulin and on intact microtubules, but was not sensitive to actin disruption or to inhibition of protein synthesis. Resealed cells were viable for days and retained the capacity to endocytose and to proliferate. The active domains of large clostridial toxins were introduced into three different cell lines. The domains were derived from Clostridium difficile B-toxin and Clostridium sordelli lethal toxin, which glycosylate small G-proteins, and from Clostridium botulinum C2 toxin, which ADP-ribosylates actin. After delivery with SLO, all three toxins disrupted the actin cytoskeleton to cause rounding up of the cells. Glucosylation assays demonstrated that G-proteins Rho and Ras were retained in the permeabilized cells and were modified by the respective toxins. Inactivation of these G-proteins resulted in reduced stimulus-dependent granule secretion, whereas ADP-ribosylation of actin by the C. botulinum C2-toxin resulted in enhanced secretion in cells. The presented method for introducing proteins into living cells should find multifaceted application in cell biology.

Insulin promotes rapid delivery of N-methyl-d- aspartate receptors to the cell surface by exocytosis

Insulin potentiates N-methyl-d-aspartate receptors (NMDARs) in neurons and Xenopus oocytes expressing recombinant NMDARs. The present study shows that insulin induced (i) an increase in channel number times open probability (nPo) in outside-out patches excised from Xenopus oocytes, with no change in mean open time, unitary conductance, or reversal potential, indicating an increase in n and/or Po; (ii) an increase in charge transfer during block of NMDA-elicited currents by the open channel blocker MK-801, indicating increased number of functional NMDARs in the cell membrane with no change in Po; and (iii) increased NR1 surface expression, as indicated by Western blot analysis of surface proteins. Botulinum neurotoxin A greatly reduced insulin potentiation, indicating that insertion of new receptors occurs via SNARE-dependent exocytosis. Thus, insulin potentiation occurs via delivery of new channels to the plasma membrane. NMDARs assembled from mutant subunits lacking all known sites of tyrosine and serine/threonine phosphorylation in their carboxyl-terminal tails exhibited robust insulin potentiation, suggesting that insulin potentiation does not require direct phosphorylation of NMDAR subunits. Because insulin and insulin receptors are localized to glutamatergic synapses in the hippocampus, insulin-regulated trafficking of NMDARs may play a role in synaptic transmission and plasticity, including long-term potentiation.

Anthrax toxin-mediated delivery of a cytotoxic T-cell epitope in vivo.

The protective antigen (PA) component of anthrax toxin mediates entry of the toxin's lethal factor (LF) and edema factor into the cytosolic compartment of mammalian cells. The amino-terminal domain of LF (LFn; 255 amino acids) binds LF to PA, and when fused to heterologous proteins, the LFn domain delivers such proteins to the cytoplasm in the presence of PA. In the current study, we fused a 9-amino acid cytotoxic T-lymphocyte (CTL) epitope (LLO91-99) from an intracellular pathogen, Listeria monocytogenes, to LFn and measured the ability of the resulting LFn-LLO91-99 fusion protein to stimulate a CTL response against the epitope in BALB/c mice. As little as 300 fmol of fusion could stimulate a response. The stimulation was PA-dependent and occurred with the peptide fused to either the amino terminus or the carboxyl terminus of LFn. Upon challenge with L. monocytogenes, mice previously injected with LFn-LLO91-99 and PA showed a reduction of colony-forming units in spleen and liver, relative to nonimmunized control mice. These results indicate that anthrax toxin may be useful as a CTL-peptide delivery system for research and medical applications.

Chronic control of high blood pressure in the spontaneously hypertensive rat by delivery of angiotensin type 1 receptor antisense.

The renin-angiotensin system plays a crucial role in the development and establishment of the hypertensive state in the spontaneously hypertensive (SH) rat. Interruption of this system's activity by pharmacological means results in the lowering of blood pressure (BP) and control of hypertension. However, such means are temporary and require the continuous use of drugs for the control of this pathophysiological state. Our objective in this investigation was to determine if a virally mediated gene-transfer approach using angiotensin type 1 receptor antisense (AT1R-AS) could be used to control hypertension on a long-term basis in the SH rat model of human essential hypertension. Injection of viral particles containing AT1R-AS (LNSV-AT1R-AS) in 5-day-old rats resulted in a lowering of BP exclusively in the SH rat and not in the Wistar Kyoto normotensive control. A maximal anti-hypertensive response of 33 +/- 5 mmHg was observed, was maintained throughout development, and still persisted 3 months after administration of LNSV-AT1R-AS. The lowering of BP was associated with the expression of AT1R-AS transcript and decreases in AT1-receptor in many peripheral angiotensin II target tissues such as mesenteric artery, adrenal gland, heart, and kidney. Attenuation of angiotensin II-stimulated physiological actions such as contraction of aortic rings and increase in BP was also observed in the LNSV-AT1R-AS-treated SH rat. These observations show that a single injection of LNSV-AT1R-AS normalizes BP in the SH rat on a long-term basis. They suggest that such a gene-transfer strategy can be successfully used to control the development of hypertension on a permanent basis.

Fused polycationic peptide mediates delivery of diphtheria toxin A chain to the cytosol in the presence of anthrax protective antigen.

The lethal factor (LF) and edema factor (EF) of anthrax toxin bind by means of their amino-terminal domains to protective antigen (PA) on the surface of toxin-sensitive cells and are translocated to the cytosol, where they act on intracellular targets. Genetically fusing the amino-terminal domain of LF (LFN; residues 1-255) to certain heterologous proteins has been shown to potentiate these proteins for PA-dependent delivery to the cytosol. We report here that short tracts of lysine, arginine, or histidine residues can also potentiate a protein for such PA-dependent delivery. Fusion of these polycationic tracts to the amino terminus of the enzymic A chain of diphtheria toxin (DTA; residues 1-193) enabled it to be translocated to the cytosol by PA and inhibit protein synthesis. The efficiency of translocation was dependent on tract length: (LFN > Lys8 > Lys6 > Lys3). Lys6 was approximately 100-fold more active than Arg6 or His6, whereas Glu6 and (SerSerGly)2 were inactive. Arg6DTA was partially degraded in cell culture, which may explain its low activity relative to that of Lys6DTA. The polycationic tracts may bind to anionic sites at the cell surface (possibly on PA), allowing the fusion proteins to be coendocytosed with PA and delivered to the endosome, where translocation to the cytosol occurs. Excess free LFN blocked the action of LFNDTA, but not of Lys6DTA. This implies that binding to the LF/EF site is not an obligatory step in translocation and suggests that the polycationic tag binds to a different site. Besides elucidating the process of translocation in anthrax toxin, these findings may aid in developing systems to deliver heterologous proteins and peptides to the cytoplasm of mammalian cells.

Eradication of large colon tumor xenografts by targeted delivery of maytansinoids.

The maytansinoid drug DM1 is 100- to 1000-fold more cytotoxic than anticancer drugs that are currently in clinical use. The immunoconjugate C242-DM1 was prepared by conjugating DM1 to the monoclonal antibody C242, which recognizes a mucin-type glycoprotein expressed to various extents by human colorectal cancers. C242-DM1 was found to be highly cytotoxic toward cultured colon cancer cells in an antigen-specific manner and showed remarkable antitumor efficacy in vivo. C242-DM1 cured mice bearing subcutaneous COLO 205 human colon tumor xenografts (tumor size at time of treatment 65-130 mm3), at doses that showed very little toxicity and were well below the maximum tolerated dose. C242-DM1 could even effect complete regressions or cures in animals with large (260- to 500-mm3) COLO 205 tumor xenografts. Further, C242-DM1 induced complete regressions of subcutaneous LoVo and HT-29 colon tumor xenografts that express the target antigen in a heterogeneous manner. C242-DM1 represents a new generation of immunoconjugates that may yet fulfill the promise of effective cancer therapy through antibody targeting of cytotoxic agents.

Real-time detection of the surface delivery of newly synthesized membrane proteins.

Newly synthesized membrane proteins travel from the Golgi complex to the cell surface in transport vesicles. We have exploited the ion channel properties of the nicotinic acetylcholine receptor (AChR) to observe in real time the constitutive delivery of newly synthesized AChR proteins to the plasma membrane in cultured muscle cells. Whole-cell voltage clamp was employed to monitor the current fluctuations induced by carbamylcholine upon the insertion into the plasma membrane of newly synthesized AChRs, following release from a 20 degrees C temperature block. We find that the transit of vesicles to the cell surface occurs within a few minutes after release of the block. The time course of electrical signals is consistent with many of the fusion events being instantaneous, although some appear to reveal the flickering of a fusion pore. AChR-containing vesicles can fuse individually or as conglomerates. Intracellular application of guanosine 5'-[gamma-thio]triphosphate inhibits the constitutive traffic of AChRs in most cells. Individual exocytotic vesicles carry between 10 and 300 AChR molecules, suggesting that AChRs may be packed extremely densely.

Rapid retroviral delivery of tetracycline-inducible genes in a single autoregulatory cassette.

We describe a single autoregulatory cassette that allows reversible induction of transgene expression in response to tetracycline (tet). This cassette contains all of the necessary components previously described by others on two separate plasmids that are introduced sequentially over a period of months [Gossen, M. & Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89, 5547-5551]. The cassette is introduced using a retrovirus, allowing transfer into cell types that are difficult to transfect. Thus, populations of thousands of cells, rather than a few clones, can be isolated and characterized within weeks. To avoid potential interference of the strong retroviral long terminal repeat enhancer and promoter elements with the function of the tet-regulated cytomegalovirus minimal promoter, the vector is self-inactivating, eliminating transcription from the long terminal repeat after infection of target cells. Tandem tet operator sequences and the cytomegalovirus minimal promoter drive expression of a bicistronic mRNA, leading to transcription of the gene of interest (lacZ) and the internal ribosome entry site controlled transactivator (Tet repressor-VP16 fusion protein). In the absence of tet, there is a progressive increase in transactivator by means of an autoregulatory loop, whereas in the presence of tet, gene expression is prevented. Northern blot, biochemical, and single cell analyses have all shown that the construct yields low basal levels of gene expression and induction of one to two orders of magnitude. Thus, the current cassette of the retroviral construct (SIN-RetroTet vector) allows rapid delivery of inducible genes and should have broad applications to cultured cells, transgenic animals, and gene therapy.