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INTRODUCTION |
Gene therapy is emerging as a promising novel therapeutic approach
to treat human disease (1-5). However, technical barriers related to
the biology of vector cell interaction and patient safety remain the
most critical end points in clinical trials. Therefore, the success of
human gene therapy depends on effective transfer and expression of the
desired genes into the target cell at the lowest possible risk for the
individual. Gene therapy approaches most commonly utilize recombinant
viral vectors, in which both cellular uptake and transgene expression
depend on interactions between the viral vector and the target cell (3,
4, 6). The best established vector system for achieving stable
transduction in human cells is the amphotropic retrovirus vector, with
its infectivity or cellular uptake at least in part determined by the
presence of natural receptors on the target cells. Since the use of
retrovirus vectors is restricted to specific receptor-expressing cells,
transduction efficiency is largely determined by cell surface receptor
density (7-9). Due to the lack of ecotropic receptors on human cells,
unmodified ecotropic retrovirus cannot be used for transduction of
human cells.
Currently, all clinical approaches for stable transduction of human
cells rely on amphotropic retrovirus. The rare event of formation of
replication-competent amphotropic retrovirus could result in systemic
viremia of a treated individual, as amphotropic retrovirus can infect
and replicate in most human cells. If technology would permit the
effective use of ecotropic retrovirus for human gene therapy protocols,
this crucial safety concern could be alleviated. Uncontrolled virus
replication in humans with ecotropic virus would be unlikely,
since the lack of natural receptors prevents infection of human
cells. Here we have developed and evaluated a strategy to accomplish
this goal by synthesizing ecotropic retrovirus molecular conjugates
(eMMLV-PL).1 To prove
feasibility for this concept, the first step would require physical or
chemical modifications of ecotropic retrovirus, not only permitting
infection of human cells but also the efficient cellular transduction
with the transferred gene. The development of strategies to overcome
ecotropic/amphotropic receptor-dependent vector uptake and
enhancement of cellular transduction efficiency through circumventing
the natural pathway of virus to cell entry has become a major goal of
gene therapy research.
We previously determined that recombinant adenovirus molecular
conglomerate vectors substantially enhanced vector uptake and transgene
expression after formation of polylysine-based conglomerates over
recombinant adenovirus particles alone (10). Here, we investigate the
hypothesis that polylysine-conjugated retrovirus results in increased
uptake and gene expression over the equivalent of unconjugated virus
(Fig. 1A). Furthermore, we
hypothesized that through this procedure, the tropism restriction of
ecotropic virus could be overcome, and human cells would become
amenable to transduction with ecotropic retrovirus vectors (Fig.
1B). If successful, this conjugate ecotropic vector would
have an additional significant safety advantage over currently employed
amphotropic systems.

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Fig. 1.
Retrovirus molecular conglomerates;
proposed mechanism. aMMLV or eMMLV encoding the lacZ
reporter gene is directly linked to polylysine via the
heterobifunctional chemical linker EDC or via biotin-avidin technology
to from retrovirus-polylysine molecular conglomerates (aMMLV-PL and
eMMLV-PL). aMMLV enters human cells via a specific receptor
(A, arrow 3). Polylysine enters cells via heparan
receptors or nonspecifically via positive charges (A,
arrow 2). The conglomerate aMMLV-PL enters the cell via the
polylysine component or via the amphotropic receptor (A,
arrows 1 and 2, respectively). eMMLV does not
infect human receptors due to a lack of ecotropic cell surface
receptors (B, arrow 3). Physically linking eMMLV
to PL (eMMLV-PL) enables cellular uptake of eMMLV to human cells
through the PL mechanism of cell entry (B, arrow
2).
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MATERIALS AND METHODS |
Cells and Culture Conditions--
The human embryonic kidney
cell 293 line was provided by Dr. Robert Gerard, University of
Texas Southwestern, Dallas, TX (also available from ATCC
((Manassas, Virginia))) (11). The human retinoblastoma cell line 911 was provided by Dr. Bout (12). The human pulmonary epithelial
carcinoma-derived cell line A549 was purchased from ATCC. All cell
lines were cultured in Dulbecco's modified Eagle's medium (Life
Technologies, Inc) supplemented with 10% fetal bovine serum (Life
Technologies, Inc.) and penicillin/streptomycin (Cellgro Mediatech,
Herndon, VA) and maintained at 37 °C under a 5% CO2
atmosphere in a humidified incubator (standard cell culture conditions). Primary human bone marrow cells were obtained by standard
aspiration from the iliac crest of healthy voluntary donors and
collected in MyeloCult H5100 medium (StemCell Technologies, Vancouver,
BC, Canada) supplemented with 250 units/ml heparin. The institutional
review board at Louisiana State University Health Sciences Center of
New Orleans, LA approved this study involving human subjects. For
erythrocyte lysis, specimens were incubated for 1 min in 0.747%
NH4Cl solution. After two washing steps with phosphate-buffered saline (PBS), 1×107 cells were seeded
in 10-cm dishes (Nunc, Naperville, IL) and cultured in Dulbecco's
modified Eagle's medium supplemented with 15% fetal bovine serum and
penicillin/streptomycin and maintained under standard conditions.
Non-adherent cells were decanted with media exchange 2-3 times/week. A
near confluent bone marrow stroma layer of fibroblast morphology was
established after 2-3 weeks. Gene transfer experiments in these cells
were performed at 70-80% confluence.
Retroviral Vectors--
The amphotropic retroviral helper cell
line PA317 or the ecotropic retroviral packaging cell line GP+E86 were
transfected with the plasmid pLZ12 using calcium phosphate (13, 14).
Plasmid pLZ12 was described by Galileo et al. (15) and is a
modification from plasmids originally made by Casadaban et
al. (16). Plasmid pLZ121 encodes the lacZ
reporter gene under control of the Rous sarcoma virus promoter and has
a 5' nuclear localization signal (Fig. 1).
The retroviral helper cell line was cultured under standard conditions
(14). Supernatants were collected from stable vector-producing cells,
and retrovirus purification was performed after a procedure previously
described by Akatsuka et al. (17). Briefly, a 30% (w/w)
stock solution of polyethylene glycol 8000 (Sigma) was prepared in
double-distilled water and stored in aliquots at 4 °C. Viral supernatants were gently mixed with polyethylene glycol in 250-ml polystyrene tubes to achieve a final 8% polyethylene glycol solution. The mixture was maintained overnight at 4 °C. After centrifugation at 1500 × g for 45 min, the precipitate was dissolved
in 3 ml of TES buffer (10 mM Tris-HCl, pH 7.2, 2 mM EDTA, 150 mM NaCl). The titer of virus
preparations was determined as previously described on NIH3T3
monolayers (18;19).
Chemical Linkage of Retrovirus to Polylysine--
Retrovirus was
covalently linked to poly-L-lysine (PL) (Sigma), molecular
mass 30-70 kDa, using the heterobifunctional chemical linker
1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)
(Pierce). Briefly, 3.6 ml of retrovirus (7 × 107
colony-forming units/ml) was resuspended in Hepes-buffered saline (HBS;
5 mM HEPES pH 7.8, 150 mM NaCl) and 0.4 ml of
PL (6 mg/ml in HBS). 0.04 ml of freshly made EDC solution (250 mg/ml in
dH2O) was added, and the reaction mixture was incubated on
ice for 4 h. This ratio of reagents was found to be optimal, as
determined in several pilot experiments in different human and murine
cell lines (data not shown). The product was then dialyzed overnight at
4 °C against a large volume of TES using 12-14-kDa cutoff membrane tubing (Spectra/Por, Laguna Hills, CA). The final product,
retrovirus-polylysine (eMMLV-PL) was separated into aliquots in TES and
stored at
80 °C.
Chemical Linkage of Avidin to Polylysine--
NeutrAvidin (NA)
(Pierce) was covalently linked to PL, molecular mass 30-70 kDa, using
the heterobifunctional chemical linker EDC. Briefly, 10 mg/ml NA was
resuspended in HBS. 1.5 ml of NA solution was gently mixed with
0.5 ml of PL (6 mg/ml in HBS) before 0.04 ml of freshly made EDC
solution (250 mg/ml in dH2O) was added. The reaction
mixture was incubated on ice for 4 h. The product was then
dialyzed overnight at 4 °C against a large volume of PBS using
12-14-kDa cutoff membrane tubing (Spectra/Por, Laguna Hills, CA). The
final product, avidin-polylysine was separated into aliquots in PBS and
stored at
20 °C.
Biotinylation of Retrovirus--
Retrovirus was covalently
linked to sulfo-N-hydroxysuccinimide-biotin (biotin)
following the instructions of the manufacturer (Pierce). Briefly, 0.2 ml of biotin (10 mg/ml in Me2SO) was mixed with 2.8 ml of
retrovirus in PBS (7×107 colony-forming units/ml). The
reaction mixture was incubated on ice for 2 h. The product
(eMMLV-B) was then exchanged in TES buffer (0.01 M
Tris-HCl, pH 7.2, 0.002 M EDTA, 0.15 M NaCl)
over EP 10 DG columns (Bio-Rad). EMMLV-B was separated into
aliquots and stored in TES at
80 °C. The titer of eMMLV-B was
determined as previously described on NIH3T3 monolayers (20).
Gene Transfer Analysis--
LacZ staining was performed as
previously described (21). Briefly, cells were fixed in 0.05%
glutaraldehyde phosphate buffer (150 mM sodium chloride, 15 mM sodium phosphate, pH 7.3) (Sigma) for 10 min at room
temperature and rinsed with PBS. Cells were stained for lacZ
expression overnight at 37 °C using freshly made X-gal solution
buffer (1 mg/ml X-gal, 20 mM potassium ferrocyanide, 20 mM potassium ferricyanide, and 2 mM
MgCl2 in PBS). After rinsing with 70% ethanol, cells were
analyzed by microscopy for blue cells.
Polymerase Chain Reactions (PCR)--
Genomic DNA was extracted
from cell pellets using a DNA extraction kit (Qiagen, Valencia, CA). A
unique and specific 1,036-base pair sequence of the Escherichia
coli lacZ gene was amplified using the primer pair
5'-GCC-GAC-CGC-ACG-CCG-CAT-CCA-GC-3' (sense) and
5'-GCG-GCG-CGG-TGA-CCA-CAC-CC-GG-3' (antisense) using Taq polymerase (Promega, Madison, WI). Amplification conditions consisted of a 1-min denaturing cycle at 94 °C, a primer annealing step at
65 °C for 1 min, and a primer extension step at 72 °C for 2 min
for a total of 35 cycles. A final elongation cycle was performed for 10 min at 72 °C (MJ Research, Peltier Thermal Cycler, Watertown, MA).
The product was analyzed after electrophoresis on a 1% agarose gel
after ethidium bromide staining under UV fluorescence.
For quantitative REAL-time PCR, a unique and specific 84-base pair
sequence of the E. coli lacZ gene was amplified
using the primer pair E. coli B-gal-f
5'-ATG-CGC-CCA-TCT-ACA-CCA-A-3' (sense) and E. coli
B-gal-rev 5'-AGA-AAC-AAC-CCG-TCG-GAT-TCT-C-3' (antisense). Product
labeling was performed using the fluorochrome SYBR® Green following
the instructions recommended by the manufacturer (PE Biosystems, Foster
City, CA). Amplification conditions consisted of a 10-min AmpliTaq Gold
polymerase activation step at 95 °C followed by a denaturing step at
95 °C for 15 s and a combined annealing and extension step at
60 °C for 1 min in a thermocycler (GeneAmp®5700 sequence detection
system, PE Applied Biosystems). A total of 40 cycles of sequential
denaturing/annealing and extension was performed. A standard curve was
established by concomitant amplification of DNA extracted from a
retrovirally stably transduced NIH3T3 clone. The samples were analyzed
with the 5700 software detection system and corrected for total DNA
content using a spectrophotometer (DU640B, Beckman Instruments). An
R of >0.99 was required for standard curves. The
sensitivity of the assay was determined to be 10 copies/sample. Copy
number per cell was determined by determining sample LacZ copy from a
standard curve.
Southern Blot Analysis--
Genomic DNA was extracted form
different lacZ-expressing 911 single cell clones previously
transduced with eMMLV-PL using a commercial DNA extraction kit (Qiagen,
Chatsworth, CA). 40 µg of restriction-digested genomic DNA and
standards established with the lacZ gene derived from the
NotI-digested control plasmid pACCMVLacZ (21) were loaded on
a 0.8% agarose gel and separated by electrophoresis following
previously described procedures (22). The DNA was transferred to
positively charged nylon membranes (MSI, Westborough, MA), which was
subsequently hybridized with a 32P-radiolabeled probe
specific for the lacZ sequence
(5'-GCC-GAC-CGC-ACG-CCG-CAT-CCA-GC-3'). After removal of nonhybridized
probe by washing, the membrane was exposed to radiographic film and
developed. Restriction digestion of pACCMVLacZ with EcoRI
linearized the plasmid, yielding a 12-kb fragment; digestion with
NotI released a 4.3-kb cassette encoding the lacZ
gene (21). For demonstration of integration by junction fragments,
genomic DNA was digested with XbaI. There is a unique XbaI site in the retrovirus construct just 3' of the
lacZ reporter gene (15, 16). The frequency of
XbaI restriction sites in human genomic is approximately 1/4
kb. For evaluation of lacZ copy number integrated into host
cells, genomic DNA was digested with XhoI and
XbaI to release a 4.4-kb fragment encoding the
lacZ gene within the retrovirus construct (15, 16). Genomic
XhoI- and XbaI-digested DNA was analyzed by
Southern blot together with different dilutions of purified
NotI-digested pACCMVLacZ plasmid DNA fragments to establish
a standard. The membranes were probed with the above-described
lacZ-specific probe, and the radiographic intensities were
determined using a chemiluminescent 4000 low light imaging system
(Alpha Innotech Corp., San Leandro, CA). Copy number per cell was
calculated using previously published formulas where the band intensity
of the transduced clones were compared with intensities from the
standard curve (22-24).
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RESULTS |
Formation of Polylysine-based Retrovirus Molecular Conglomerates
(aMMLV-PL) Results in Enhanced Gene Expression--
To test the
hypothesis that formation of polylysine-based retrovirus molecular
conglomerates would improve transduction efficiency over retrovirus
particles, biotinylated aMMLV encoding the lacZ reporter
gene (aMMLV-B) and avidinylated polylysine (PL-NA) were synthesized.
The biotinylation procedure did not significantly reduce the
infectivity and transgene expression of aMMLV-B over unmodified virus
(aMMLV) in murine NIH3T3 cells (data not shown).
Human 911 cells were infected with aMMLV-B with or without Polybrene at
increasing m.o.i. (0.05-1.5) or the equivalent amount of aMMLV-B as
molecular conglomerate by adding PL-NA (aMMLV-PL). Formation of
retrovirus conglomerates significantly increased transgene expression
at low m.o.i. by up to 5-fold. At higher m.o.i., transduction
efficiency reached 100% for all vectors as expected (Fig.
2A). To investigate the
ubiquity of this transduction enhancement, other cell lines, human 293, A549, and murine NIH3T3 cells, were infected with aMMLV-B (with or
without Polybrene) or the equivalent amount of retrovirus as aMMLV-PL.
At low m.o.i. (0.2), formation of aMMLV-PL resulted in significant,
5-fold-increased transgene expression in the examined cell lines (Fig.
2B).

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Fig. 2.
Enhanced gene transfer by formation of
amphotropic retrovirus conglomerates (aMMLV-PL) to human and murine
cell lines. Human 911 cells were infected using different
amphotropic retrovirus constructs: biotinylated
lacZ-encoding amphotropic retrovirus (aMMLV-B),
aMMLV-B with Polybrene (Pbre), and aMMLV-B with avidinylated
polylysine (PL-NA) as complete retrovirus conglomerates
(aMMLV-PL). Near confluent cell monolayers were incubated
with different constructs consisting of equivalent m.o.i. of aMMLV-B as
shown beneath the x axis (0.05-1.5). Cells were analyzed
for lacZ transgene expression by X-gal staining, and the
percentage of positive (blue) cells is indicated on the y
axis (A). Human 293, 549, and murine NIH3T3 cells were
incubated with aMMLV-B, aMMLV-B with Polybrene, and aMMLV-B with PL-NA
at the m.o.i. of 0.2 per construct and analyzed for lacZ
transgene expression (B). Each bar represents the
mean of triplicates + SE. The asterisks indicate
a p value of 0.05 or less. Depicted results are
representative and consistent for a series of four independent
experiments.
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Formation of Polylysine-based Retrovirus Molecular Conglomerates
(eMMLV-PL) Overcomes Tropism Restriction of Ecotropic
Retrovirus--
To study the hypothesis that formation of
polylysine-based conglomerates overcome the tropism restriction of
ecotropic retroviruses, biotinylated eMMLV encoding the lacZ
reporter gene (eMMLV-B) was joined to PL-NA to form eMMLV-PL. Similar
to aMMLV-B, the biotin modification did not significantly decrease
infectivity of eMMLV (data not shown). Human embryonal kidney 293 cells
were infected with different vector and control constructs at an m.o.i.
of 0.4 or 1.5 and analyzed for reporter gene expression. As expected, no transgene expression was observed with unmodified ecotropic virus
(eMMLV) or eMMLV-B. However, physical linkage of eMMLV-B to PL-NA with
conglomerate formation of eMMLV-PL resulted in significant transgene
expression of 25 and 84%, respectively (Fig.
3).

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Fig. 3.
Gene transfer to human 293 cells with
ecotropic retrovirus through formation of ecotropic retrovirus
polylysine conglomerates. lacZ-encoding ecotropic
retrovirus (eMMLV), PL, and different modifications thereof were
synthesized using biotin-avidin technology. Near confluent human 293 cell monolayers were incubated with different constructs and compounds
as shown beneath the x axis. Equivalent m.o.i. of eMMLV (0.4 and 1.5) were used for each group. Cells were analyzed for
lacZ transgene expression by X-gal staining and, the
percentage of positive (blue) cells is indicated on the y
axis. Depicted results are representative and consistent for a series
of four independent experiments. Pbre, Polybrene;
ND, not determined.
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Polylysine-based Ecotropic Retrovirus Molecular Conglomerates
(eMMLV-PL) Transduce Various Human Cell Lines and Primary Human Bone
Marrow Stroma Cells at High Efficiency--
The human retinoblastoma
cell 911, the human lung cancer cell line A549, and freshly isolated
primary human bone marrow-derived adherent stroma cells were
transfected with eMMLV or eMMLV-PL at the m.o.i. of 0.5 and 1.5. Cells
were not cultured under selective conditions favoring proliferation of
transduced cells. Transgene expression was only seen in cells
transfected with eMMLV-PL with 32 and 87% for 911 cells, 25 and 82%
for A549 cells, and 30 and 92% for primary human stroma cells (Figs.
4 and
5).

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Fig. 4.
Formation of retrovirus molecular
conglomerates overcomes ecotropic tropism restriction in different
human cell lines and primary human cells. Human cell lines 911, A549, and primary human bone (BM) marrow-derived stroma
cells were incubated with 0.4 or 1.5 m.o.i. of lacZ
encoding eMMLV or the equivalent virus as molecular conjugate
(eMMLV-PL). Cells were analyzed for lacZ transgene
expression by X-gal staining, and the percentage of positive (blue)
cells is shown on the y axis. Each bar represents
the mean of triplicates ±SE. The asterisks indicate a
p value of 0.05 or less. Depicted results are representative
and consistent for a series of four independent experiments.
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Fig. 5.
Transgene expression in human 293 and primary
bone marrow stroma cells after eMMLV-PL lacZ gene
transfer. Human 293 cells (A and B) and
primary human bone marrow stroma cells (C and D)
were transfected with eMMLV or eMMLV-PL, respectively. Cells were
analyzed for lacZ transgene expression as outlined under
"Materials and Methods" and visualized by transmission light
microscopy.
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Retrovirus Molecular Conglomerates (eMMLV-PL) Integrate into the
Genome of Human Cells at One Copy per Cell--
Integration of
lacZ gene into the host genome was demonstrated by genomic
DNA restriction digestion of five 911 single cell clones using the
enzyme XbaI. Different sized junction fragments were
identified in all clones examined for the presence of lacZ (Fig.
6). The copy number of the transferred
reporter gene in transduced 911 cells was measured by REAL-time
PCR and by Southern blot. A copy number of 0.91/cell (0.87-0.95) was
quantified by PCR, and a copy number of 0.83/cell was quantified by
Southern blot (Fig. 7, A and
B). The combined evidence for these experiments indicates
that eMMLV-PL results in integration of the transgene at 1 copy/cell.

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Fig. 6.
Ecotropic retrovirus molecular conjugate
(eMMLV-PL) gene transfer to human cells results in genomic transgene
integration. After transduction of 911 cells with eMMLV-PL,
lacZ-positive single cell clones cells were established.
Genomic DNA was restriction-digested with the enzyme XbaI, a
single cutter within the retrovirus genome. For positive
control, the plasmid pACCMVLacZ was NotI-digested
to release the 4.3-kb lacZ cassette. The DNA was then
analyzed by Southern Blot using a probe specific for the
lacZ gene.
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Fig. 7.
Determination of transgene copy number
after retrovirus molecular conjugates (eMMLV-PL) gene transfer.
After transduction of 911 cells with eMMLV-PL, lacZ-positive
single cell clone cells were established. REAL-time PCR was
performed using a specific probe for lacZ on two clones
(denoted by triangles and squares) and compared
to a standard curve established with the lacZ-encoding
plasmid pACCMVLacZ (dots). Cycle threshold
(CT) values are plotted against lacZ copy
number (A). For quantitative Southern blot analysis, genomic
DNA was restriction-digested with XhoI and XbaI
to release the 4.4-kb lacZ cassette from the integrated
retrovirus construct. Plasmid pACCMVLacZ was restriction-digested with
the enzyme NotI, releasing the 4.3-kb lacZ gene
to establish a standard. The membrane was hybridized using a probe
specific for the lacZ gene as outlined under Materials and
Methods." Signal intensity was used to calculate copy number per cell
(B).
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DISCUSSION |
Our findings demonstrate that ecotropic retrovirus could be
modified with a conjugate technique to accomplish effective
transduction of human cells. If successful, this procedure could
potentially provide additional safety features for use in humans.
Ecotropic virus is chemically linked within the conjugate, so it
remains genetically unaltered. The theoretical risk of in
vivo propagation is substantially less for a transient tropism
modification compared with a genetically tropism-modified ecotropic
retrovirus vector such as vesicular stomatitis virus-pseudotyped
envelopes. In the event that transiently modified ecotropic virus
becomes replication competent, its progeny would be ecotropic
retrovirus, which is incapable of propagating in humans. On the other
hand, progeny arising from a genetically engineered, tropism-modified
virus could continue to maintain its tropism-modified form and, thus, would remain infectious to human cells, posing a significant safety risk.
First, we had to proof that the polylysine conjugation does indeed
enhance the transduction efficiency of retrovirus. Although previous
studies showed in principle feasibility of retrovirus retargeting via
alternate cell surface receptors, several retargeting studies were
associated with significant reduction or even disappearance of cellular
transduction (25) (26, 27, 28). In contrast, greatly enhanced
transduction efficiency was reported for retrovirus vectors redirected
by pseudotyping with the vesicular stomatitis virus envelope (29,
30).
To investigate the hypothesis that polylysine conjugation would enhance
retrovirus transduction, retrovirus molecular conglomerates were
synthesized by biotinylation and linking to avidinylated polylysine.
Our data demonstrate that at low m.o.i., formation of molecular
conjugates synthesized with amphotropic virus (aMMLV-PL) substantially
enhances transgene expression over the equivalent amount of free
amphotropic retrovirus (aMMLV-B). The human cell lines tested were
permissive to infection with amphotropic retrovirus; thus, increasing
m.o.i. eventually resulted in nearly 100% transduction efficiency with
both virus vectors. Therefore, the advantage in this particular
experimental setting was limited to a transduction enhancement observed
at low m.o.i.
The mechanism of the increased transduction efficiency of amphotropic
conjugates could be explained through enhancement of virus uptake via
their natural receptors or the utilization of an alternate pathway for
virus uptake. The former concept has been proposed for
fibronectin-meditated retrovirus transduction augmentation (31, 32).
This model proposes that fibronectin facilitates colocalization of
retrovirus particles and target cells with a large increase in local
virus titer presented to the cell (31, 33, 34). Similarly, the adjunct
use of the positively charged polycations such as Polybrene in
retroviral gene transfer procedures has long been known to
nonspecifically promote retrovirus transduction efficiency and is now
used for many protocols (35).
To proof that the entry mechanisms of retrovirus-polylysine
conjugates are independent of natural receptors and are entirely polylysine-mediated, experiments were designed using ecotropic retrovirus constructs in human cells. We found that only the physical binding of eMMLV to polylysine accomplished cellular transduction with
efficiencies of 80-90% in different human cell lines and primary
cells. Although polycations have previously been reported to facilitate
infectivity and to enhance transduction efficiency (36), our data
indicate that this effect can not only be greatly enhanced by chemical
or physical linkage of virus and polycation, but this linkage allows an
ability to completely overcome receptor-dependent virus
uptake. This phenomenon could be explained by the ability of positively
charged polylysine to nonspecifically penetrate cell surface membranes
and enter the cytosol (37, 38). Compared with individually retargeted
retrovirus particles, conglomerates consist of multiple virus particles
bound to polylysine, thus providing a higher likelihood of delivering
one intact virus to the nucleus. This explanation of an added survival
benefit is supported by studies where DNA bound to polylysine was found
to have significantly enhanced resistance to intracellular degradation over free DNA (39). Similar to DNA, the retrovirus particles may be
relatively protected from nuclease digestion within the polylysine
conglomerate formation. Therefore, the improved transduction efficiency
could result as the combined effect of both polylysine-mediated enhanced uptake and also improved retrovirus survival from cytosolic degradation.
Transduction with eMMLV-PL in human cells resulted in a similar genomic
copy number as eMMLV in the murine cell line NIH3T3. This implies that
the formation of retrovirus conglomerates does not change the
fundamental principal established for retrovirus/cell interactions of
only a single integration event with one retroviral copy per cell
(40).
In summary, this novel retrovirus-based gene transfer system has the
potential to improve transduction efficiency that comes from low
receptor expression, such as for instance in hematopoietic cells (8,
9). Moreover, this system would provide additional safety features over
amphotropic retrovirus vectors currently used in human trials. Even the
case of formation of replication-competent ecotropic virus would
be theoretically a self-limited problem, as progeny of transiently
modified ecotropic virus could not further propagate in humans due to
its tropism restriction. Last, the polylysine-based retrovirus system
may enable specific receptor retargeting by formation of
receptor-targeted retrovirus molecular conglomerate vectors (10,
41).