WO2001044450A1

WO2001044450A1 - Enhancing trehalose biosynthesis

Info

Publication number: WO2001044450A1
Application number: PCT/US2000/042198
Authority: WO
Inventors: Fred Levine
Original assignee: University of California Berkeley; University of California San Diego UCSD
Current assignee: University of California Berkeley; University of California San Diego UCSD
Priority date: 1999-11-16
Filing date: 2000-11-16
Publication date: 2001-06-21
Anticipated expiration: 2002-05-16
Also published as: AU4507001A

Abstract

The present invention provides a vector encoding trehalose-6-phosphate synthase (otsA)/trehalose-6-phosphate phosphatase (otsB) fusion protein and methods by which the fusion protein may be expressed in mammalian cells to provide enhanced desiccation tolerance. In a preferred embodiment, the otsA/B fusion protein is subcloned into CMV forming a bicistronic cassette for transfection. Preferentially, a short linker sequence connects the otsA gene 5' to the otsB gene so that, when expressed, the fusion protein maintains the ability to fold in correct confirmation. In another preferred embodiment, the fusion protein can be further subcloned into recombinant adenovirus conferring trehalose expression to a wider variety of cell types.

Description

ENHANCING TREHALOSE BIOSYNTHESIS

This application claims the benefit of priority of United States Provisional Application Serial No. 60/1 65,799 to Fred Levine, filed November

1 6, 1 999 and entitled ENHANCING TREHALOSE BIOSYNTHESIS, the entire contents of which is incorporated herein by reference.

FIELD OF THE INVENTION This invention generally relates to a fusion protein encoding trehalose expression, and more specifically, to the expression of the E. colt trehalose biosynthetic genes in mammalian cells.

BACKGROUND OF THE INVENTION A variety of unicellular and multicellular prokaryotic and eukaryotic organisms, including certain bacteria, plants and insects, are able to survive for long periods of time in a dehydrated or desiccated state in which they contain as little as 0.1 % water by weight. These organisms, which may be classified as "anhydrobiotic" or "cryptobiotic", include Streptomyces sp. spores, dry active bakers yeast, brine shrimp cysts, some species of adult and larval nematodes, a pre-pupil larvae of one species of sawfly, and at least one plant species, Selaginella lepidophylla. The ability of these organisms to tolerate such harsh conditions has to do with their ability to synthesize the non-reducing disaccharide trehalose ( 1 -α-D-glucopyranosyl- 1 , 1 -α-D-glucopyranoside) using naturally occurring cellular biosynthetic mechanisms. The action by which trehalose mediates this protective effect is not well understood but involves a complex mechanism, possibly affecting both lipids within the cell membrane, as well as proteins.

Trehalose is synthesized from uridine diphosphate glucose and glucose-6-phosphate. The first step of this process, resulting in the formation of trehalose-6-phosphate, is catalyzed by trehalose-6-phosphate synthase, encoded by the otsA gene in E. coli. In the second step of the synthetic process, the dephosphorylation of trehalose-6-phosphate is catalyzed by trehalose-6-phosphate phosphatase, encoded by the ots gene in E. coli. These genes are present in the otsBA operon in E. coli.

The currently accepted explanation for the desiccation-protective effects of trehalose is based on the fact that trehalose is able to depress the temperature at which desiccating lipid membranes undergo phase transition from a gel to a liquid-crystal state. When membranes which are rehydrated below the phase transition temperature, they undergo phase transition. This transition causes damage to the membrane, resulting in a loss of membrane integrity and cell death. Rehydration above the phase transitions temperature does not alter membrane integrity. It is thought that, through retention of the "glassy" liquid-crystal state, damage to cellular membranes is minimized and membrane leakage is prevented upon rehydration of the organism, thereby preserving cellular viability. Trehalose has been shown to inhibit protein denaturation by exclusion of water from the protein surface in the hydrated state. By maintaining proteins in their native state, aggregation is inhibited during heat stress. This preserves the structure of proteins in the dry state, probably by replacing surface water molecules that contribute to the maintenance of properly folded protein structure.

In addition to improving the ability of lower organisms to withstand desiccation, trehalose has also been shown to enhance the preservation of mammalian cells and tissues. The ability to desiccate and store mammalian cells, including those from humans, would greatly simplify the storage and transportation of cells and possibly organs. For trehalose to be used to enhance desiccation tolerance, it must be expressed intracellularly as the trehalose molecule is not capable of readily crossing cellular membranes.

Current preservation protocols most often involve the exogenous addition of trehalose in addition to other cryopreservation agents to the cell or tissue requiring preservation. In contrast, the present invention provides for the introduction of trehalose biosynthetic machinery into mammalian cells by way of a vector conferring enhanced levels of expression of the trehalose biosynthetic genes. The ability to dry and rehydrate cells and complex multicellular structures will have great implications for the use of many kinds of tissue engineered products.

SUMMARY OF THE INVENTION The present invention provides a vector encoding trehalose-6- phosphate synthase (o.sA)/trehalose-6-phosphate phosphatase (otsB) fusion protein and methods by which the fusion protein may be expressed in mammalian cells to provide enhanced desiccation tolerance. In a preferred embodiment, the otsA/B fusion protein is subcloned into CMV forming a bicistronic cassette for transfection. Preferentially, a short linker sequence connects the otsA gene 5' to the otsB gene so that when expressed, the fusion protein maintains the ability to fold in correct confirmation. In another preferred embodiment, the fusion protein can be further subcloned into recombinant adenovirus conferring trehalose expression to a wider variety of cell types.

The present invention also provides a method by which desiccation tolerance may be conferred to a cell through the introduction of an adenoviral vector containing a fusion protein encoding trehalose-6-phosphate synthase (otsA)/ trehalose-6-phosphate phosphatase (ofsB). The adenoviral vector containing the CMV-OTS expression cassette is cloned and expressed in 1 2F human primary foreskin fibroblasts. The presence or absence of trehalose production is detectable using standard HPLC measurement means.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference is now made to the following detailed description of the embodiments illustrated in the accompanying drawings, wherein:

Figure 1 illustrates the biosynthetic pathway in which trehalose is synthesized from uridine diphosphate glucose;

Figure 2 shows a model of trehalose production within a mammalian cell. Figure 2a depicts trehalose production mediated by individual otsA and otsB gene products. Figure 2b depicts trehalose production mediated by an otsAlB fusion gene product where the otsA and otsB gene products are bound by a flexible linker;

Figure 3 illustrates the CMV -ots Al otsB bicistronic expression cassette;

Figure 4 illustrates the CMV-oteA/B fusion gene expression vector;

Figure 5 is a plot illustrating the multiplicity of infection (MOI) vs. time for trehalose production in 1 2F cells infected with either: Figure 5a, Ad-OTS or Figure 5b, Ad-OTS and Ad-GFP;

Figure 6 is a graph representing viability of dried and rehydrated 1 2F cells infected with Ad-OTS at MOI of 200, 400 and 800;

Figure 7 is an HPLC analysis of trehalose. Figure 7a shows the HPLC analysis of a control sample without trehalose. Figure 7b shows the HPLC analysis of a purified trehalose standard. Figure 7c shows the HPLC analysis of an extract of 293 cells transfected with pCMV/OTS. Figure 7d shows the HPLC analysis of an extract of 293 cells transfected with pCMV/OTS treated with trehalose for two hours; and

Figure 8 depicts FTIR spectroscopy readouts of hydrated and desiccated 1 2F cells. Figure 8a is the FTIR spectroscopic analysis measuring water content of three separate samples of hydrated 1 2F cells. Figure 8b is a magnified view of Figure 8a to better visualize the sample peaks.

DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS

In Escherichia coli, trehalose biosynthesis is controlled at the genetic level by the otsAIB locus. This locus encodes trehalose-6-phosphate synthase (otsA), which catalyzes the synthesis of trehalose-6-phosphate from UDP-glucose and glucose-6-phosphate, and trehalose-6-phosphate phosphatase (otsB) which catalyzes the formation of trehalose. Figure 1 shows the biosynthetic pathway for which trehalose is the final product. The coding region of the ofsA gene is 474 amino acids in length (Seq. No. 1 ), while the coding region of the otsB gene is 266 amino acids in length (Seq. No. 2). The otsB gene, which begins with a valine rather than a methionine, as for most starting codons, is 3' of the otsA gene with an overlap of 25 base pairs.

The most rational approach to increasing the efficiency with which trehalose-6-phosphate is converted to trehalose is to increase the concentration of trehalose-6-phosphate in close proximity to the trehalose-6- phosphate enzyme. In yeast, for example, the trehalose biosynthetic machinery exists as a complex between the trehalose-6-phosphate synthase, trehalose-6-phosphate phosphatase, and a 1 23kDa regulatory subunit. The function of this subunit seems to involve holding together the trehalose synthase complex, and conferring sensitivity to physiological concentrations of phosphate and to fructose-6-phosphate. However, there is currently no data as to whether the E. coli otsA and otsB proteins form a similar complex as in yeast. Current evidence does suggest that the otsA and ofsB gene products locate randomly in mammalian cells, however this leads to inefficient conversion of trehalose-6-phosphate to trehalose. As such, no vertebrate has been shown to be capable of synthesizing trehalose or exhibiting the degree of desiccation tolerance observed in organisms able to manufacture trehalose. Nevertheless, the exogenous addition of trehalose has been shown to be effective in the ex vivo storage and cryopreservation of mammalian cells and organs in the hydrated state.

The rationale behind the present invention is based on the premise that bringing trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase into close proximity by expression of an otsA/B fusion protein could increase the local concentration of trehalose-6-phosphate in the vicinity of the trehalose-6-phosphate phosphatase activity thereby increasing the efficiency of conversion to trehalose. Figure 2 is a model of trehalose production within a mammalian cell. Figure 2a depicts trehalose production mediated by individual otsA and otsB gene products, as occurs most often in native vertebrate cells. The otsA and otsB gene products are not co- localized, resulting in inefficient conversion of trehalose-6-phosphate and therefore low levels of trehalose. In contrast, Figure 2b depicts trehalose production mediated by an otsA/B fusion gene product, as in the present invention, where the otsA and otsB gene products are bound by a flexible linker. Increased local concentrations of trehalose-6-phosphate lead to a more efficient conversion to trehalose, resulting in lower overall trehalose-6- phosphate levels and increased levels of trehalose.

In the method of the present invention, the otsA and otsB genes are cloned using known standard PCR protocols, from the E. coli strain DH5α utilizing the published sequence of the E. coli ots operon as a guide for the design of the PCR primers. Further modifications are made to enhance translational initiation in eukaryotic cells as well as to replace valine with a methionine at the start of the ofsB gene. DNA sequence analysis of the cloned genes shows a single base pair divergence from the published sequence. This mutation occurs in the third position of a codon and does not result in an alteration of the individual amino acid or the amino acid sequence. To determine the ability of the newly manufactured ots genes to mediate trehalose production in mammalian cells, the otsA and otsB genes can be inserted into an expression vector driven by the cytomegalovirus early promoter (CMV). Translation of the otsB protein in the CMV driven vector occurs through the use of the internal ribosome entry site from poliovirus

(PO) polyadenylation (PA) sequence. Figure 3 is a diagrammatic representation of the CMV-otsA/otsB bicistronic expression cassette.

To express a trehalose-6-phosphate synthase/ trehalose-6-phosphate phosphatase fusion protein, the otsA and otsB genes are fused with the ofsA gene 5' of the otsB gene and the stop codon of the otsA gene deleted. In order to maximize the likelihood that the fusion gene will maintain proper spatial conformity, a short linker comprising four glycines and two serines is inserted between the otsA and otsB genes, allowing the two enzymes to fold correctly. The amino acid composition of the linker allows for maximal flexibility and resistance to proteases, and interferes minimally with protein folding. Once it is established that the otsA and otsB genes are properly and adequately fused to each other via the flexible linker and that the fusion construct is functional, the construct can be cloned into an expression vector driven by the CMV promoter. Figure 4 is a diagrammatic representation of the otsA/B fusion gene expression vector as inserted into the CMV promoter and given the designation pCMVofsA/B (OTS).

Expression of trehalose in mammalian cells In order to determine whether trehalose can be expressed and detected in mammalian cells, 293 cells are transfected with the pCMV-OTS construct. The human embryonic kidney cell line 293 was obtained from the American Type Culture Collection. All cells in this and following studies are grown in Dulbecco's Modified Eagle's Medium (DMEM; Gibco Laboratories) supplemented with 1 0% fetal bovine serum (FBS; Gibco Laboratories).

48 hours following transfection, the cells are extracted and analyzed using standard HPLC technique for the presence of trehalose. As shown in Figure 7a, HPLC analysis of un-transfected 293 cells demonstrates the absence of a peak at 28 minutes retention time, which corresponds to trehalose as is evident of the HPLC analysis of a purified trehalose standard, shown in Figure 7b. HPLC analysis of 293 cells transfected with the pCMV-

OTS construct shows a large peak at 28 minutes retention time (corresponding to trehalose) as can be seen in Figure 7c when compared to the trehalose standard of Figure 7b. To confirm that the obvious peak at 28 minutes retention time observed for the transfected 293 cells actually represents the presence of trehalose, the cell extract was incubated with trehalose enzyme, resulting in an almost complete disappearance of the putative trehalose peak, as is shown in Figure 7d. Analysis of these data may be construed as meaning the peak identified at 28 minutes represents the presence of trehalose at a concentration of 1 .0-1 .5 nM in the 293 cells transfected with pCMV-OTS at a multiplicity of infection (MOI) of 10⁶.

Development and characterization of an adenoviral vector expressing otsA and otsB

For trehalose to be expressed in a wider variety of cell types, the CMV-OTS expression cassette of the present invention is inserted into an adenoviral vector and given the designation Ad-OTS. Additionally, an adenoviral vector expressing green fluorescent protein, designated Ad-GFP, is constructed to be used as a parallel control for characterizing expression of the vectors. Having constructed the Ad-OTS and Ad-GFP adenoviral vectors, they are used to infect 1 2F human primary foreskin fibroblasts at multiplicity of infection (MOI) of 1 00, 500 and 1 000 pfu/cell. The human primary foreskin fibroblast cell line 1 2F was provided by Advanced Tissue

Sciences. Standard HPLC analysis of extracts of the infected 1 2F cells indicate the presence of trehalose in cells infected with Ad-OTS but not in cells infected with Ad-GFP. Figure 5a shows the direct relationship between the amount of trehalose produced in the infected cells versus the MOI. Maximal levels of trehalose production ranged from 1 .0-1 .5 nM/1 0⁶ cells at an MOI of 1000 PFU/cell, the highest MOI tested.

Trehalose expression in mammalian cells is non-toxic Intracellular accumulations of trehalose-6-phosphate are toxic to E. coli and most likely other cell types. It is important to determine whether trehalose, expressed in mammalian cells through transfection using the fusion protein adenoviral cassette of the present invention, results in cellular toxicity. To do this, the viability of cells infected with the Ad-OTS expression vector and Ad-GFP, as a control, are compared. 1 2F cells are seeded at a density of 3x10⁵ cells per well in a standard six-well culture plate. Sixteen hours following the initial cell seeding, the cells are infected with either Ad-OTS or Ad-GFP viral particles at MOIs ranging from 100 to 1 000 PFU/cell. One day following infection, growth medium is replaced with fresh media and the cultures are incubated for an additional 24 hours.

Adherent cells (cells remaining adhered to the surface of the well following removal of growth medium) are then harvested and stained with calcein AM (CAM) to visualize live cells and ethidium homodimer-1 (EthD-1 ) to visualize dead cells (Live/Dead Viability/Cytotoxicity Kit; Molecular Probes). Cells simultaneously exhibiting green granular perinuclear staining and red nuclear staining are interpreted as being in the process of dying and are counted as dead. At high MOIs, toxicity was observed in cells infected with both the Ad-OTS and Ad-GFP vectors, as is commonly seen with adenoviral vectors, shown in the graph of Figure 5b. No detectable difference between the Ad- OTS and Ad-GFP viruses is observed, therefore demonstrating that trehalose produced using the expression vector of the present invention is non-toxic for mammalian cells.

Human cells containing trehalose can be dried and rehydrated with maintenance of a high degree of viability

To determine whether mammalian cells expressing trehalose demonstrate improved desiccation tolerance, 1 2F cells are seeded in six-well culture plates at a density of 3x10⁵ cells per well. Three hours post seeding, cells are infected in triplicate with either Ad-OTS expressing the fusion gene of the present invention or Ad-GFP as a control, at MOIs ranging from 200 to 800 PFU/cell. Drying of the samples is accomplished by complete removal of the tissue culture medium seventy-two hours after infection, followed by isolation of each culture plate in a sealed plastic bag, then stored at room temperature. For the purpose of determining whether the dried cells have retained viability, fresh tissue culture medium is added to selected cell samples following varying times in the dry state. Viability of the rehydrated cell samples is determined by calcein AM/ ethidium homodimer-1 (EthD-1 ) staining as previously described above.

Following 24 hours in the desiccated state, 1 2F cells infected with Ad- OTS retain a high degree of viability while cells infected with the Ad-GFP control are completely dead as determined through photomicrographic analysis (not shown) of the cell samples. However, the viability of the Ad- OTS infected cells declines as the length of time in the desiccated state is extended, as is shown by the plot of cell viability versus time of Figure 6. While viable cells are sometimes partially recoverable after a period of five days in the desiccated state, consistent retention of viable cells is achievable after three full days of desiccation. There is significant variability in the optimal MOI for retention of maximal viability, ranging from 200-800 PFU/cell. This may be attributable to variation in infection efficiency depending on the state of the cells at the time of infection combined with a balance between maximizing trehalose production and minimizing toxicity from adenoviral infection.

Dried human cells do not contain detectable water For the purpose of explaining the observed decrease in viability of dried cells over time, Applicants seeks to demonstrate that the cells maintained without tissue culture medium for 24 hours still contain enough moisture to lead to a loss of viability, while other cell samples, maintained without tissue culture medium for longer periods of time, contain less water. Using Fourier transform infrared spectroscopy (FTIR), the water content of dried 1 2F cells can be measured after 24, 48 and 72 hours of drying.

To measure water content, the region of the water spectrum associated with bond stretching modes (both symmetric and asymmetric), occurring nominally at 3600-3800 cm^"1 in the IR spectrum, is used. Preliminary studies with a number of different materials compatible with cell growth demonstrate that glass coverslips, used in sample preparation, have a consistently low IR absorbence in the above designated region, making them an ideal substrate for measuring the water content of cells grown on a two-dimensional surface. As such, 1 2F cells are grown on glass coverslips placed in six-well culture plates (3x10⁵ cells/well) and consequently infected with Ad-OTS at varying MOIs. 24, 48 and 72 hours post-infection, all tissue culture medium is removed from the respective wells. The six-well plates with sample cultures are subsequently sealed with PARAFILM™ and stored at room temperature for either 24, 48 or 72 hours. To distinguish between IR signals arising from water and from other cellular components that may have IR absorbence in the 3000-3700 cm^"1 range, controls are prepared in which confluent and subconfluent 1 2F cells grown on glass coverslips are baked at 80 °C overnight to remove all available water within the sample.

Under the room temperature drying conditions specified above for preparation of the samples, drying of the cell samples occurs extremely rapidly. Additionally, three control samples dried at room temperature, exhibit a rapid loss of water content within the approximately 5 minutes of being removed from the wells containing tissue culture medium to being serially analyzed by FTIR. The FTIR spectra are recorded at room temperature in absorbance mode on a Prospect-IR FTIR spectrometer (Midac Corp., Irvine, CA) operating with GRAMS/32 (Galactic Industries, Salem, NH) utilizing a mid-IR spectral region from 400-4000 cm^'1 (spectra are composed of 1 868 points, collected by averaging 1 6 scans having a resolution of 4 cm^" ¹). Cell samples having been dried at room temperature show no detectable water content compared with like samples having been oven baked overnight. Figure 8a shows FTIR analysis of three cell samples containing hydrated 1 2F cells that have been removed simultaneously from wells containing tissue culture medium. Water content is measured sequentially by FTIR over a period of five minutes for both "wet" cells and cells dried at room temperature and stored at room temperature for 24, 48 or 72 hours.

Additionally, samples of confluent (cells grown to confluence) and subconfluent (cells grown to approximately 50% confluent) cells baked at 80 °C overnight, as controls, are included in the FTIR measurement. Figure 8b is a magnification of the FTIR analysis, excluding two of the "wet" cell samples, to better visualize the areas containing the water peak in the dried samples. The IR absorbance curves for the samples in which the cells have been dried for 24, 48 or 72 hours fall between the two curves representing confluent and subconfluent control samples dried at 80 °C overnight. This can be interpreted as thus indicating that the water content for the 24, 48 and 72 hour cell samples has reached the minimum possible value. Any remaining IR signal at 3300 cm^"1 in the baked control samples may be due either to water that is so tightly bound that it can not be removed from the cells or to other cellular components that absorb in a similar IR range. Nevertheless, it is demonstrated that little or no water remaining in the dried cell samples having been infected with the fusion gene product of the present invention retain a high degree of viability upon rehydration.

Through application of the present invention, the production of trehalose in human cells confers the ability to remain viable for several days in the absence of water. Previous studies have shown that trehalose can confer desiccation protection to lower organisms (Crowe, et al.; Annual Review of Physiology 54, 579-99; 1992) by protecting multiple different cellular components, including membrane components (Crowe, et al. Developments in Biological Standardization 74, 285-94; 1992)(Sun, et al. Biophysical Journal 70, 1 769-76; 1996), and proteins (Allison, et al. Archives of Biochemistry and Biophysics 365, 289-98; 1999), (Leslie, et al.

Applied and Environmental Microbiology 61 , 3592-7; 1995), (Crowe, et al. Biochemical Journal 242, 1 -1 0; 1987). However, it had not been known whether it was possible to provide desiccation protection for higher organisms merely by inducing intracellular biosynthesis of trehalose. In the method of the present invention, the trehalose biosynthetic gene for E. coli is delivered to primary fibroblasts using a recombinant adenovirus, and in doing so demonstrating that trehalose may be synthesized in human cells to a degree sufficient to confer desiccation protection. In many organisms, the trehalose biosynthetic machinery exists in a complex in which the two biosynthetic enzymes, otsA and otsB, are associated with other subunits that play a regulatory role in trehalose biosynthesis (Thevelein, et al.; Trends in Biochemical Sciences 20, 3-10; 1995). The method of the present invention demonstrates that the expression of a fusion protein in which otsA and otsB are connected by a flexible linker and inserted into an adenovirus is sufficient to result in high levels of trehalose biosynthesis and that such nucleated mammalian cells can be reversibly desiccated. Such a method can be adapted to preserve mammalian cells that are part of complex, multicellular structures such as organs. Advances in gene transfer technology and optimization of the process of desiccation and storage are likely to result in major improvements on the length of time that viability can be maintained in the dry state.

The following examples demonstrate the utility and method of the present invention with the understanding that these particular examples in no way limit the scope of the invention. EXAMPLE 1 : CLONING OF E. COLI otsA AND ofsB

To develop a mammalian expression vector for the ofsA and ofsB genes, a 1 .4 kb fragment (Seq. ID. No. 1 ) encoding ofsA (trehalose-6- phosphate synthase) and a 0.8 kb fragment (Seq. ID. No. 2) encoding ofsB (trehalose-6-phosphate phosphatase) were amplified from E. coli DH5α cells using standard PCR protocols. A bicistronic unit comprising these two genes was created in the cloning vector pGEM^®-7z( + ) (Promega Corp., Madison Wl.) utilizing the poliovirus internal ribosome entry sequence as described by Pelletier, et al. (Nature 334, 320-325; 1988). The resulting o.sA-PO-ofsB fragment was subsequently subcloned into a CMV expression plasmid pCMV-MNK to generate the plasmid containing otsAIB fusion gene pCMV- OTS.

EXAMPLE 2: RECOMBINANT ADENOVIRUS VECTORS A BstXI / BstXI restriction fragment from pCMV-OTS, extending from

5' of the CMV promoter to 3' of the polyadenylation sequence was subcloned into the EcoRV site of the adenoviral vector shuttle plasmid pXCX2 using protocols previously described by Spessot, et al. ( Virology 1 68,

378-87; 1989). The resulting plasmid, pXCX2/CVM-OTS, was used along with the adenovirus plasmid pJM 1 7 to generate recombinant adenovirus by techniques known in the art (Berkner, et al. ; Current Topics in Microbiology and Immunology 1 58,39-66; 1991 ). Recombinant adenovirus expressing green fluorescent protein (GFP) gene under control of the chicken β-actin promoter was used as a control as described by Leibowitz, et al. (Diabetes 48, 745-753; 1999).

EXAMPLE 3: TREHALOSE ASSAY BY HPLC

2x1 0⁶ of infected or uninfected 1 2F cells were pelleted by centrifugation, resuspended in 1 ml of distilled water, lysed using standard freeze-thaw technique and then centrifuged to remove extraneous cellular debris. The resulting supernatant was distributed into two aliquots. 1 00 μl of 1 35 mM citric acid, pH 5.7 was added to each aliquot. For trehalose digestion, 0.03 units of dialyzed trehalose enzyme was added and the samples were incubated at 37 °C for 2 hours, followed by boiling to inactivate the trehalose enzyme. For samples which were to remain undigested by trehalose enzyme, the same volume of 25 mM potassium phosphate was added in place of trehalose enzyme. 5 nM trehalose in water was used as a standard for the HPLC analysis and as a digestion control for trehalose-treated samples. Following centrifugation at 5,000g for 1 0 minutes, the resulting supernatant was loaded on Micro Bio-Spin Chromatography columns (Bio-Rad) with mixed bed analytical grade ion exchange resin to eliminate charged particles, as trehalose is a neutral sugar. Bio-Spin column flow-through was dried using a Savant Speed-Vac concentrator. 200 μ\ of distilled water was added to each sample for HPLC analysis.

HPLC analysis was done using a Dionex system DX-500, w/ AS3500 equipped with a CarboPac MA1 , 4x250 mm column (Dionex) at a flow rate of 0.4 ml/minute at 8 °C. An ED 40 electrochemical detector was used to quantitate the amount of trehalose. Water and 1 M sodium hydroxide were used as eluents. Elution was performed using a sodium hydroxide gradient

. After loading, the HPLC column was washed with 200 mM sodium hydroxide for 40 minutes followed by a 200-660 mM continuing gradient over the course of 25 minutes.

EXAMPLE 4: EFFECT OF TREHALOSE AND VACUUM ON DESICCATION

TOLERANCE OF F1 2 CELLS

F1 2 cells were plated at 85% confluence in 5 - six well plates. On plates designated 1 , 3 and 5, trehalose (500 mM final concentration in growth media) was introduced by thermal shock. The media was removed the following day. Plates 1 , 2 and 5 were vacuum sealed and maintained at room temperature. Plates 3 and 4 were sealed in bags with no vacuum and also maintained at room temperature. Fresh media was added to plates 1 -4 at four days later. The following day, cells were harvested and stained with propidium iodide (PI) and Calcein/Ethidium. At seven days post, the cells from plate 5 were harvested in similar fashion. A dead/alive assay as previously described was performed by fluorescence activated cell sorter analysis (FACS). The following Tables 1 and 2 quantify the finding.

Table 1 Calcein/Ethidium staining/FACS

Table 2 PI staining/FACS

Other embodiments and modifications of the present invention may occur to those of ordinary skill in the art in view of these teachings. Therefore, this invention is to be limited only by the following claims which include all other such embodiments and modifications when viewed in conjunction with the above specification and accompanying drawings.

Claims

What is claimed is:

1 . A recombinant gene construct comprising coding sequence for a fusion protein encoding E. coli trehalose biosynthetic enzymes selected from the group consisting of trehalose-6-phosphate synthase and trehalose- 6-phosphate phosphatase.

2. The recombinant gene construct of claim 1 , wherein the fusion protein coding sequence comprises: a gene encoding otsA (trehalose-6-phosphate synthase) (Seq. ID. No.1 ); a gene encoding otsB (trehalose-6-phosphate phosphatase) (Seq. ID. No. 2); and a flexible amino acid linker coding sequence.

3. The recombinant gene construct of claim 1 , wherein the fusion protein coding sequence is subcloned into CMV, said subclone forming a bicistronic cassette for transfection.

4. The recombinant gene construct of claim 2, wherein the amino acid linker coding sequence comprises six amino acids.

5. The recombinant gene construct of claim 4, wherein the amino acid linker coding sequence comprises four glycine residues and two serine residues.

6. The recombinant gene construct of claim 2, wherein the otsA gene is positioned 5' to the otsB gene.

7. The recombinant gene construct of claim 2, wherein the amino acid linker coding sequence conjoins the otsA gene to the otsB gene.

8. The recombinant gene construct of claim 3, wherein the subclone forming the bicistronic cassette is further subcloned into adenovirus.

9. A method for providing desiccation tolerance to a cell comprising inserting into said cell an adenoviral vector containing a fusion protein encoding trehalose-6-phosphate synthase (otsA) and trehalose-6- phosphate phosphatase (otsB).

10. The method of claim 9, wherein the fusion protein provides desiccation tolerance to a cell and comprises: a gene encoding otsA (trehalose-6-phosphate synthase) (Seq. ID. No.1 ); a gene encoding otsB (trehalose-6-phosphate phosphatase) (Seq. ID. No. 2); and a flexible amino acid linker coding sequence.

1 1 . The method of claim 9, wherein desiccation tolerance is provided to a cell, said cell being a mammalian cell.