JOURNAL OF VIROLOGY, Dec. 1996, p. 8459–8467 Vol. 70, No.

12
0022-538X/96/$04.0010
Copyright q 1996, American Society for Microbiology

Molecular Characterization of Replication-Competent Variants

of Adenovirus Vectors and Genome Modifications
To Prevent Their Occurrence
KATHLEEN M. HEHIR, DONNA ARMENTANO, LISA M. CARDOZA, TERRY L. CHOQUETTE,
PATRICIA B. BERTHELETTE, GARY A. WHITE, LARRY A. COUTURE,† MARIA B. EVERTON,
JESSE KEEGAN, JOHN M. MARTIN, DENISE A. PRATT, MICHAEL P. SMITH,
ALAN E. SMITH, AND SAMUEL C. WADSWORTH*
Genzyme Corporation, Framingham, Massachusetts 01701-9322
Received 17 May 1996/Accepted 19 August 1996

Adenovirus (Ad) vectors for gene therapy are made replication defective by deletion of E1 region genes. For
isolation, propagation, and large-scale production of such vectors, E1 functions are supplied in trans from a
stable cell line. Virtually all Ad vectors used for clinical studies are produced in the 293 cell, a human
embryonic kidney cell line expressing E1 functions from an integrated segment of the left end of the Ad type
5 (Ad5) genome. Replication-competent vector variants that have regained E1 sequences have been observed
within populations of Ad vectors grown on 293 cells. These replication-competent variants presumably result
from recombination between vector and 293 cell Ad5 sequences. We have developed Ad2-based vectors and have
characterized at the molecular level examples of replication-competent variants. All such variants analyzed are
Ad2-Ad5 chimeras in which the 293 cell Ad5 E1 sequences have become incorporated into the viral genome by
legitimate recombination events. A map of Ad5 sequences within the 293 cell genome developed in parallel is
consistent with the proposed recombination events. To provide a convenient vector production system that
circumvents the generation of replication-competent variants, we have modified the Ad2 vector backbone by
deleting or rearranging the protein IX coding region normally present downstream from the E1 region such
that the frequency of recombination between vector and 293 cell Ad5 sequences is greatly reduced. Twelve serial
passages of an Ad2 vector lacking the protein IX gene were carried out without generating replication-
competent variants. In the course of producing and testing more than 30 large-scale preparations of vectors
lacking the protein IX gene or having a rearranged protein IX gene, only three examples of replication-
competent variants were observed. Use of these genome modifications allows use of conventional 293 cells for
production of large-scale preparations of Ad-based vectors lacking replication-competent variants.

Gene therapy vectors based on viruses require modifications
that eliminate their disease-causing potential during therapeu-
tic use. This is normally achieved by disabling their ability to
replicate independently in patients. A parallel and somewhat
counter requirement is that viral vectors retain the ability to
replicate in a producer cell such that large quantities of vector
can be manufactured. This is accomplished most commonly by
deleting a subset of sequences from the viral genome and
placing these sequences within a producer cell in which they
function in trans. As a consequence of these conflicting re-
quirements, viral gene transfer vectors are almost invariably
compromised in their growth properties. Thus, genetic variants
with improved growth properties that might arise during vector
propagation will have strong selective advantages compared
with the parent vector and if unchecked can represent a sig-
nificant proportion of vector preparations. Of particular con-
cern with regard to safety is the emergence of variants that
have reacquired the ability for replication independent of the
producer cell, so-called replication-competent viruses (RCV).
Such RCV may be able to replicate in patients with unknown
but potentially deleterious effects. A likely mechanism for the
* Corresponding author. Mailing address: Genzyme Corporation,
1 Mountain Rd., Framingham, MA 01701-9322. Phone: (508) 872-
8400. Fax: (508) 872-9080. Electronic mail address: SCW@World.std
.com.
† Present address: Ribozyme Pharmaceuticals, Inc., Boulder, CO
80301.
generation of RCV is through recombination between vector
and producer cell sequences.
For retrovirus gene therapy vectors, the potential for the
presence of replication-competent retroviruses is a significant
concern because of the oncogenic potential of the parent vi-
ruses. In fact, oncogenic variants of murine leukemia virus
arising within gene therapy vector preparations have been ob-
served (5, 16) and shown to cause disease in monkeys. Replica-
tion-defective adenovirus (Ad)-based vectors, especially those
based on Ad serotypes 2 and 5 (Ad2 and Ad5), are attractive
candidates for gene therapy because of the modest disease
potential of the parental viruses. To date, Ad-based vectors
used clinically have been made replication defective through
deletion of early region 1 (E1) genes (reviewed in reference 8).
Such vectors have been produced exclusively in the human 293
cell line, which contains a segment of the Ad5 genome that
supplies the E1 functions necessary for vector growth in trans
(8, 9). Given the structure of E1-deleted Ad vectors and their
growth on 293 cells, the potential exists for generation of rep-
lication-competent Ad (RCA) in vector preparations, and
Lochmüller et al. (13) have reported the isolation of RCA.
Despite the relatively low pathogenicity of the parent Ad vec-
tors, the potential risk associated with the presence of RCA in
clinical Ad vector preparations should be assessed for several
reasons. First, it has not been formally proven that RCA arise
through recombination with Ad5 sequences in the 293 cell
genome, thus leaving their origin in doubt. Second, the struc-
tures of individual RCA isolates have not been investigated at

8459
8460 HEHIR ET AL. J. VIROL.

FIG. 1. Ad2-based CFTR gene transfer vectors. The vectors Ad2/CFTR-1 and Ad2/CFTR-2 have been described previously (2, 14). Construction of the vectors
Ad2/CFTR-3 and Ad2/CFTR-6 is described in Materials and Methods. The identical CFTR cDNA (2, 14) is encoded by each of the vectors, but different combinations
of promoter and poly(A) addition signal sequences are used as indicated. The E1 region deletion endpoints in each vector are indicated by the numbered hash marks.
The Ad2/CFTR-1 vector has a wild-type E4 region, while the E4 region in each of the other vectors has been replaced by E4 ORF6 coding sequences (2). PGK,
phosphoglycerate kinase; BGH, bovine growth hormone.

the molecular level. Third, the frequency of occurrence of
RCA has not been examined in detail. Finally, the impact of
contaminating RCA with regard to safety of Ad vector prep-
arations and in interpretation of preclinical studies has not
been investigated.
We have been developing Ad2-based vectors encoding the
cystic fibrosis (CF) transmembrane conductance regulator
(CFTR) protein for treatment of CF. Our first vector, Ad2/
CFTR-1, was a simple E1 replacement vector in which tran-
scription of the CFTR cDNA was controlled by the endoge-
nous E1A promoter (14). Because the CFTR cDNA is large,
4.5 kb, and because we chose to leave the E3 region intact, the
genome of Ad2/CFTR-1 is approximately 104.5% of the wild-
type Ad2 genome (14). The safety and efficacy of Ad2/CFTR-1
for CFTR gene transfer was analyzed extensively in vitro and
in vivo in rodents and nonhuman primates and was shown to
correct transiently the CFTR chloride secretion defect in the
nasal respiratory epithelium of CF patients (14, 17, 18). How-
ever, despite its encouraging safety and efficacy profile, Ad2/
CFTR-1 is difficult to produce in large quantities because it has
relatively poor growth properties. As a consequence, Ad/
CFTR-1 is easily overgrown by RCA or by mutants in which
sequences not required for growth in vitro have been deleted.
These shortcomings of Ad/CFTR-1 led to the development of
a second-generation vector, Ad2/CFTR-2, in which the vector
genome size was reduced through a partial deletion of the E4
region (2).
Because of the unknown safety hazards of RCA and the lack
of detailed information about RCA structure, we undertook a
detailed study of this problem. We describe here the molecular
structures of RCA isolates from a series of Ad2/CFTR vectors
and a model for how such recombinants arise by passage
through the 293 cell. These molecular analyses were possible
because 293 cells contain Ad5 sequences whereas the vectors
are based on Ad2. We also describe modifications to the basic
vector backbone that greatly reduce the frequency of RCA
occurrence.
MATERIALS AND METHODS
Construction of recombinant Ad2 vectors. Except for Ad2/CFTR-1, each of
the vectors used in this study is based on the Ad2/CFTR-2 vector, in which the
E4 region has been replaced by the open reading frame 6 (ORF6) cDNA and the
phosphoglycerate kinase promoter is used to control transcription of the CFTR
cDNA (2) (Fig. 1). The vectors Ad2/CFTR-3 and Ad2/CFTR-6 utilize the Ad2
E1A promoter to control transcription of the CFTR cDNA. Additional differ-
ences are that a truncated bovine growth hormone polyadenylation signal se-
quence (2) is used in Ad2/CFTR-3 whereas the simian virus 40 (SV40) polyad-
enylation signal sequence is used in Ad2/CFTR-6.
In Ad2/CFTR-7, the CFTR expression cassette is similar to that present in
Ad2/CFTR-6 in that the Ad2 E1A promoter and SV40 polyadenylation signal
sequence are used. However, the E1 region deletion in this vector was extended
from nucleotide 3328 to nucleotide 4019 to delete the protein IX (pIX) coding
sequences. A portion of the E3B region between nucleotides 29293 and 30840
was deleted in Ad2/CFTR-7. Deletion of E3 sequences was carried out to reduce
the genome size of this vector to 95% of the wild-type genome size and thereby
allow packaging in the absence of pIX (4, 6).
In Ad2/CFTR-8, the CFTR expression cassette and pIX deletion are identical
to those in Ad2/CFTR-7. However, pIX promoter and coding sequences (nucle-
otides 3519 to 4061) were cloned downstream from the ORF6 cDNA. An SV40
VOL. 70, 1996 REPLICATION-COMPETENT ADENOVIRUS STRUCTURE
polyadenylation signal sequence was also cloned into a position between the
ORF6 and pIX sequences. The pIX gene in this vector is transcribed in a right-
to-left direction.
All vectors were propagated in 293 cells and purified by CsCl centrifugation as
previously described (14).
Restriction endonuclease mapping of 293 cell Ad5 sequences. High-molecular-
weight DNA from 293 cells was digested with the indicated restriction endo-
nucleases and analyzed by agarose gel electrophoresis and Southern blot hybrid-
ization. Restriction endonuclease cleavage sites were mapped to the left or right
of the E1 region by hybridization with a probe containing sequences to the left
of the SphI site at position 3663 or with the adjacent SphI fragment probe.
RCA assay. The RCA assay was carried out by infecting HeLa cells with the
test dose of vector for 4 days and then passaging a freeze-thaw lysate of the HeLa
cell culture on to cultures of A549 cells. HeLa cells are used in the first step
because they can withstand Ad vector cytotoxicity up to a multiplicity of infection
(MOI) of 200. Under similar conditions, A549 cells exhibit severe vector induced
cytotoxicity that masks the presence of RCA contamination. During the second,
longer step of the assay, RCA plaques can be observed readily on A549 cells but
not on HeLa cells.
The HeLa cell phase of the RCA assay is carried out in either 10-cm-diameter
dishes or roller bottles, depending on the size of the test dose. In either case, the
vector MOI is limited to ,25 to minimize cytotoxicity. In the 10-cm-diameter
plate assay, vector is added to HeLa cells in 10 ml of medium per plate and is left
on the culture for the 4-day culture period. In the A549 cell phase of the assay,
the HeLa cell lysate is applied in 10 ml and left on the culture for the entire
10-day culture period. The A549 cultures are fed two times during the assay by
the addition of 10 ml of medium at each time. In the roller bottle RCA assay,
vector is added to HeLa cells in 100 ml of medium per bottle and is left on the
culture for the 4-day culture period. The HeLa cell lysate is applied to A549 cells
in a volume of 100 ml per bottle, which is left on the culture for 3 days. The roller
bottle cultures are fed over the next 9 days at least twice by replacement of half
of the medium with fresh medium. The observation of either individual plaques
or widespread cytopathic effect (CPE) was taken as an indication of the presence
of RCA in the initial test dose. Parallel assays of the same vector doses mixed
with known amounts of wild-type Ad2 were carried out to ensure that the vast
excess of replication-defective vector in the original inoculum did not mask the
presence of RCA.
Analysis of RCA genomes. Viral DNA from each isolate was prepared from a
Hirt extract of infected A549 cells and was analyzed first by restriction endonu-
clease cleavage using BclI and BamHI. Subsegments of the RCA genomes
between the left-hand inverted terminal repeat and position 5219 were amplified
by PCR and sequenced. The sequences of the RCA genomes were then com-
pared with the Ad2 and Ad5 sequences.
Blind passage protocol. A stock of each vector designated passage 0 was
established with an inoculum of 10 infectious units (IU). Passage 1 was initiated
at an MOI of 5, and subsequent passages were initiated at an estimated MOI of
5. Vector titers were measured at either passage 6 or 7 and passage 12. RCA
assays were carried out as described above at passages 3, 6, and 12. Vector doses
of 1.25 3 108, 2.5 3 109, and 2 3 1010 IU were tested, with the MOI in the HeLa
cell phase of the assay being ,25. The assay was scored as a pass if no CPE was
detected or a fail if either extensive CPE or individual plaques were detected.
Thermostability determinations. Samples of CsCl-purified preparations of
Ad2/CFTR-2, Ad2/CFTR-7, and Ad2/CFTR-8 were incubated in microcentri-
fuge tubes in a water bath at 378C for the indicated times. The samples were
frozen at 2808C until the residual vector titers were determined. Titers for each
samples were determined in duplicate, and the average of the two values was
graphed. Vector titers were determined as previously described (14).
RESULTS
Genetic variants of Ad2/CFTR-1. Ad2/CFTR-1 is an E1 re-
placement vector based on Ad2 (14). In this vector, viral se-
quences between nucleotides 545 and 3498 are replaced with
the CFTR cDNA but all other viral genes are intact. Ad2/
CFTR-1 has relatively poor growth characteristics, and as a
consequence, populations of the vector are susceptible to over-
growth by faster-growing variants. Two different classes of vari-
ants were detected in preparations of the Ad2/CFTR-1 vector:
deletions within the CFTR expression cassette (2) and recom-
binants with 293 cell Ad5 E1 sequences.
In preparation for clinical use of Ad2/CFTR-1, bioassays
based on viral CPE in cells that were nonpermissive for growth
of E1-deleted adenovirus were carried out to test for the pres-
ence of RCA. This assay readily detected RCA in an early Ad2/
CFTR-1 vector stock and in vector lots made from the stock.
To understand better the origins of RCA variants, one RCA
plaque was isolated from a preparation of Ad2/CFTR-1 and
8461
FIG. 2. Restriction endonuclease map of an RCA isolate from Ad2/CFTR-1.
An RCA arising during growth of the vector Ad2/CFTR-1 was isolated and
digested with BclI. From the digestion pattern, a map of the BclI sites in the RCA
was constructed and compared with the BclI maps of the starting vector and Ad5,
the source of E1 sequences present in the 293 cell genome. The positions of BclI
sites are indicated by the vertical hash marks. The approximate limits of the Ad5
sequences in the 293 cell are indicated by the double-headed arrow beneath the
Ad5 map.
characterized at the molecular level. Restriction endonuclease
mapping of the left end of the vector DNA revealed the pres-
ence of Ad5-specific BclI sites, suggesting that the RCA isolate
had acquired its independent growth properties through re-
combination with Ad5 sequences, presumably those contained
within the 293 cell genome. As shown in Fig. 2, the restriction
map of this RCA isolate is consistent with that of an Ad2-Ad5
chimera with Ad5 sequences present at the extreme left end of
the genome. The first Ad2-specific restriction endonuclease
site mapped in this chimera is the BclI site at Ad2 nucleotide
position 4057, with the remainder of the genome apparently
being derived from the Ad2/CFTR-1 vector. To provide more
detailed information about the structure of this chimeric ge-
nome, the region between the inverted terminal repeat and the
E1A promoter was amplified by PCR and sequenced. The
sequence between positions 22 and 352 matched the Ad5 se-
quence, confirming that the left end of the RCA genome was
derived from Ad5. These results indicate that this RCA isolate
was generated by at least one recombination event between
293 cell Ad5 sequences and Ad2/CFTR-1 and that this event
took place between nucleotide positions 3498 and 4057 of the
Ad2 genome.
RCA from a second-generation Ad2/CFTR vector. A second-
generation vector, Ad2/CFTR-2 (Fig. 1), was constructed to
overcome the problems associated with production of Ad2/
CFTR-1. The genome of Ad2/CFTR-2 was shortened to 101.3%
of the wild-type Ad2 genome by deletion of all E4 region
sequences except those encoding E4 ORF6 (2). Additional
changes in this vector compared with Ad2/CFTR-1 are the use
of the phosphoglycerate kinase promoter instead of the E1A
promoter and the use of a shortened version of the bovine
growth hormone polyadenylation signal sequence downstream
from the CFTR cDNA. With these changes, yields of the Ad2/
CFTR-2 vector on a per-cell basis are similar to those attained
with wild-type Ad2 (2).
In an earlier version of the RCA assay in which a dose of 5 3
108 IU of vector could be tested reliably, no RCA were de-
tected in the Ad2/CFTR-2 seed stock. Similarly, RCA were not
detected in numerous high-titer Ad2/CFTR-2 vector lots pro-
duced from this seed stock and tested in the same assay. How-
ever for treatment of CF, a vector dose of at least 5 3 109 IU
would be required to achieve an MOI of 1 for respiratory
epithelial cells within the lung. Therefore, more sensitive bio-
assays were developed to allow testing of higher vector doses of
Ad2/CFTR-2. More than 30 Ad2/CFTR-2 vector preparations
were assayed at a dose of 2.5 3 109 IU, and 66% were positive
for RCA. More than 20 Ad2/CFTR-2 vector preparations were
assayed at a dose 2 3 1010 IU, and all were positive for RCA.
However, no deletion mutants were detected in any prepara-
tions of Ad2/CFTR-2, in contrast to the results obtained with
Ad2/CFTR-1 (2). These results indicated that despite the vig-
8462 HEHIR ET AL. J. VIROL.

orous growth properties to Ad2/CFTR-2, RCA could still be

propagated in vector preparations at levels that could be de-
tected with a sensitive bioassay.
Molecular analysis of RCA isolates from Ad2/CFTR-2. Sev-
eral Ad2/CFTR-2 RCA isolates were analyzed at the molecu-
lar level. Restriction endonuclease mapping experiments using
BclI and ClaI indicated that each of the RCA isolates was an
Ad2-Ad5 chimera containing the partial E4 deletion diagnostic
of the parent vector and E1 region sequences of Ad5 origin
(data not shown). Two independent Ad2/CFTR-2 RCA iso-
lates, 33 and 45, were selected for nucleotide sequence analysis
over the region from the left terminus to position 5219. In
these RCA isolates, the E1 region sequences corresponding to
those deleted from the vector were exclusively of Ad5 origin
(data not shown). Within the region analyzed to the left and
right of the E1 region, there are 22 nucleotide sequence dif-
ferences between Ad2 and Ad5. These diagnostic sequence
differences allowed mapping of the general region where re-
combination took place between Ad2 vector sequences and
293 cell Ad5 sequences.
A comparison of the sequence of Ad2/CFTR-2 RCA isolate
33 with Ad2 and Ad5 sequences from positions 150 to 350 is
shown in Fig. 3A. The first four diagnostic positions in Ad2/
CFTR-2 RCA 33 all match the Ad2 sequence while the next
three diagnostic positions all match the Ad5 sequence, sug-
gesting that recombination between Ad2/CFTR-2 and Ad5
sequences occurred between positions 199 and 258. Beyond
position 3328, the right-hand E1 deletion point in the Ad2/
CFTR-2 vector, diagnostic differences between Ad2 and Ad5
exist, and all match the Ad5 sequence up to position 4056 (Fig.
3B). The next diagnostic position at 4844 matches the Ad2
sequence, indicating that recombination between Ad2/CFTR-2
and Ad5 sequences occurred between positions 4056 and 4844.
Thus, it appears that RCA isolate 33 was generated by one
recombination event to the left of the CFTR cDNA and a
second recombination event to the right of the cDNA. These
two events resulted in the reciprocal deletion of the CFTR
expression cassette and incorporation of the Ad5 E1 region
and, as a consequence, the restoration of independent growth
properties to the RCA isolate. Ad2/CFTR-2 RCA isolate 45,
obtained from a different vector production lot than isolate 33,
had the identical nucleotide sequence over the region analyzed
(Fig. 3B).
Additional vectors have been produced in our laboratory
as variants of Ad2/CFTR-1 (Fig. 1). Ad2/CFTR-3 and Ad2/
CFTR-6 utilize the endogenous E1 promoter and are based on
the second-generation partial E4 deletion backbone used to
construct Ad2/CFTR-2. The only difference between Ad2/
CFTR-3 and Ad2/CFTR-6 is that a shortened version of the
bovine growth hormone polyadenylation signal sequence is
used in the former whereas the SV40 early polyadenylation
signal sequence is used in the latter. With genome sizes of
100% (Ad2/CFTR-3) and 100.5% (Ad2/CFTR-6) of the wild-
type Ad2 genome size, these vectors would be expected to have
good growth properties. Analysis of numerous high-titer prep-
arations of these vectors indicates that this is indeed the case.
Considering the structures of Ad2/CFTR-3 and Ad2/CFTR-6
and of the RCA isolates derived from Ad2/CFTR-2, recombi-
nation between these vectors and 293 cell Ad5 sequences
would be predicted to occur. In fact, RCA have been isolated
from high-titer preparations of Ad2/CFTR-3 and Ad2/CFTR-
6, and molecular analyses of these RCA (summarized in Fig.
3B) again provided evidence of Ad2/Ad5 chimeras derived by
recombination events flanking the CFTR cDNA.
Mapping of Ad5 sequences in the 293 cell genome. Previous
mapping studies concluded that approximately 12% of the left
FIG. 3. Nucleotide sequence analysis of RCA isolates from the Ad2/CFTR
vectors. The region of the Ad genome between nucleotides 1 and 5500 from each
of the RCA isolates was amplified by PCR and sequenced directly. Shown in
panel A is a portion of the nucleotide sequence analysis of an RCA isolate from
lot 33 of the Ad2/CFTR-2 vector. A sequence comparison of Ad2 and Ad5 over
the region from nucleotides 151 to 350 is shown, with the diagnostic sequence
differences between Ad2 and Ad5 enclosed in boxes. The sequence determined
from the RCA isolate at each of these diagnostic positions is indicated by the
number within the box. The first diagnostic nucleotide position that matches the
Ad5 sequence is 258. It can be seen that the next two diagnostic nucleotide
positions also match the Ad5 sequence. The sequence determined from this
RCA isolate matches the Ad5 sequence at least through position 4056 (Ad5
numbering) but matches the Ad2 sequence at the next diagnostic position, 4853
(data not shown). This result is shown diagrammatically in panel B as are the
results of the sequence analyses of the other RCA isolates. In panel B, regions
of the RCA genomes derived from Ad2 are indicated by open boxes and those
derived from Ad5 are indicated by shaded boxes. The numbered positions above
each vector indicate the first and last sequence matches at a diagnostic position
with the Ad5 sequence, while the numbered positions below each vector indicate
the next upstream and downstream positions matching the Ad2 sequence. The
recombination events leading to the generation of the RCA isolates are pre-
sumed to have taken place between these positions.
end of the Ad5 genome, or approximately 4,300 bp, was pres-
ent in the 293 cell genome (1, 15). Our analyses of RCA iso-
lates indicated that sequences from near the left terminus of
Ad5 as well as sequences beyond position 4066 were present
within the 293 cell genome. To obtain additional information
beyond this point about the Ad5 sequences in the 293 cell
genome, we carried out mapping studies using known Ad5
restriction endonuclease sites and Ad5 E1 region hybridization
probes (Fig. 4). The right-most Ad5 restriction endonuclease
site that was found to be present within 293 cell DNA is the
BsmFI site at position 4137. The next restriction endonuclease
site that was not detected was the BsrI site at position 4280.
These analyses were complicated by the presence of hybridiz-
ing restriction fragments that were not expected from the Ad5
map and/or by restriction fragments that exhibited less than
expected levels of hybridization probe binding. For example,
only a single BsmFI fragment of 2,738 nucleotides should have
been detected with the probe used, but at least two smaller
hybridizing fragment were observed (Fig. 4B). The presence of
Ad5 DNA segments of unexpected lengths suggests that some
VOL. 70, 1996 REPLICATION-COMPETENT ADENOVIRUS STRUCTURE
of the Ad5 sequences within the 293 cell genome may be
rearranged. From the results of the analyses of RCA genomes
and the restriction mapping experiments, we conclude that the
contiguous Ad5 sequences within the 293 genome extend from
near the left terminus to at least nucleotide position 4137 but
are likely truncated soon thereafter.
Vector backbone changes to reduce recombination with 293
cell Ad5 sequences. The demonstration of recombination be-
tween Ad2 vector and 293 cell Ad5 sequences indicated that
the 293 cell is not optimal for propagation of simple E1 re-
placement vectors. One of two different strategies could be
followed to resolve this problem: construction of new producer
cell lines bearing no homology to vector genomes or construc-
tion of new vectors with minimal homology to the 293 cell Ad5
sequences. Because of the proven utility of the 293 cell for
growth of high-titer vector preparations, alteration of the Ad
genomic DNA seemed to represent the logical approach.
Mapping of the Ad5 sequences in the 293 cell genome in this
study indicated the presence of contiguous Ad5 sequences
8463
FIG. 4. (A) Contiguous Ad5 sequences present in 293 cells. 293 cell DNA
was cleaved with a variety of restriction endonucleases and analyzed by Southern
blot hybridization as described in Materials and Methods. Hybridization probes
were the Ad2 SphI fragment containing nucleotides (nt) 1 to 3663 or the Ad2
SphI fragment containing nucleotides 3664 to 5143. The thinner solid lines
indicate 293 cell genomic DNA. The patterned boxes represent the indicated
regions of the Ad5 genome. Only the positions of the BsmFI (4137) and BsrI
(4280) sites are shown. (B) Representative Southern blot hybridization result
with BsmFI- and BsrI-digested 293 cell DNA and the Ad2 SphI fragment probe
extending from nucleotides 3664 to 5143. The expected BsmFI fragment and the
position where the BsrI fragment would be expected to appear are indicated by
labeled arrows. The results of these analyses indicate that the BsmFI site at
position 4137 in the Ad5 genome is present in Ad5 293 cell DNA and that the
BsrI site at position 4354 in the Ad5 genome is absent in Ad5 293 cell DNA.
from near the left terminus to approximately position 4300. To
the left of the transgene in a typical E1 replacement vector,
there is at least 350 nucleotides of sequence homology between
vector and 293 cell Ad5 sequences, virtually all of which must
be retained for optimal vector packaging (10). If the E1 pro-
moter is retained, an additional 200 nucleotides of homologous
sequences remains between vector and 293 cell Ad5 DNA. To
the right of the transgene, the most commonly used right-hand
E1 deletion endpoint is at position 3498, resulting in an addi-
tional approximately 800 bp of nearly perfect homology be-
tween Ad2- or Ad5-based vectors and 293 cell Ad5 sequences.
The gene encoding pIX, a virion structural protein that is
dispensable for growth and packaging of viral genomes less
than 95% of wild-type length (4, 6), comprises approximately
500 bp of this region. Beyond the pIX gene lies the major late
promoter on the top strand of the virus and the IVA2 gene on
the bottom strand of the virus, genomic regions that must be
retained for efficient vector growth.
On the basis of these considerations, we constructed a new
vector, Ad2/CFTR-7, in which pIX sequences were deleted to
reduce the potential for recombination between vector and 293
cell sequences and thus reduce RCA formation. Because of the
large size of the CFTR cDNA and the requirement to keep the
genome size to less than 95% of that of wild-type Ad2, deletion
of pIX sequences within Ad2/CFTR-7 required the parallel
deletion of additional vector sequences. We chose to delete a
portion of E3B sequences because these are not required for
virus growth in vitro (Fig. 5). Ad2/CFTR-7 has growth char-
acteristics similar to those of Ad2/CFTR-2 and expresses
CFTR channel activity in polarized epithelial cell monolayers
(data not shown). Five high-titer Ad2/CFTR-7 vector prepa-
rations have been produced to date and have been tested for
the presence of RCA. No evidence of RCA contamination was
detected in these preparations in test doses of 2.5 3 109 and
2 3 1010 IU. However, after testing a total of 1011 IU in an
extended bioassay format, we detected two RCA plaques. One
isolate was recovered and shown by restriction endonuclease
mapping to be an Ad2-Ad5 chimera bearing the E3 deletion
from Ad2/CFTR-7. Thus, the deletion of pIX sequences in an
8464 HEHIR ET AL. J. VIROL.

FIG. 5. Ad2/CFTR gene transfer vectors with rearranged pIX sequences. Construction of the vectors Ad2/CFTR-7 and Ad2/CFTR-8 is described in Materials and
Methods. Ad2/CFTR-7 contains a full pIX deletion, the same partial E4 deletion as in the Ad2/CFTR-2 vector (Fig. 1), and a partial E3 deletion (29293 to 30840).
The genome length of this vector is approximately 95% of the wild-type genome length. In the Ad2/CFTR-8 vector, the pIX sequences (3519 to 4061) have been moved
to a position downstream from the ORF6 cDNA.

Ad2/CFTR vector allows large-scale production of high-titer
vector with very low levels of RCA.
Rate of RCA generation. To provide additional information
about the rate of generation of RCA in pIX-containing vectors
and to attempt to promote the generation of RCA in Ad2/
CFTR-7, a blind passage experiment was carried out. In this
experiment, the Ad2/CFTR-1, Ad2/CFTR-2, Ad2/CFTR-6,
and Ad2/CFTR-7 vectors were subjected to 12 sequential pas-
sages, each at an MOI of approximately 5. At passages 3, 6, 9,
and 12, RCA assays on increasing vector doses were carried
out, the results of which are shown in Table 1.
No RCA were detected from Ad2/CFTR-7 even after 12
passages despite the fact that this vector appeared to grow less
well in this experiment. This result confirms the utility of the
pIX deletion in reducing RCA generation. At the earliest pas-
sage tested, passage 3, RCA were detected from Ad2/CFTR-1
but were not detected from the other vectors. Restriction en-
donuclease mapping results of RCA isolates from passage 3
were consistent with an Ad2-Ad5 chimera with the E1 region
derived from Ad5. This result is consistent with existing data
on RCA isolated from Ad2/CFTR-1 and Ad2/CFTR-2. Both
Ad2/CFTR-2 and Ad2/CFTR-6 gave rise to RCA by passage
12, but there appeared to be quantitative differences in the
RCA content of these vector populations. RCA were detected
in the Ad2/CFTR-2 passage 12 population at the 2.5 3 109 test
dose, while at the 2 3 1010 test dose, widespread CPE was
observed in the A549 cell monolayer used in the test. In con-
trast, no RCA were detected in the 2.5 3 109 test dose of
Ad2/CFTR-6, and only a few RCA plaques were detected at
the 2 3 1010 test dose. These results are consistent with the fact
that Ad2/CFTR-6 has a smaller genome size and better growth
properties than Ad2/CFTR-2.
An unexpected result was obtained from the analysis of
passage 12 Ad2/CFTR-1 in that no RCA were detected. To
investigate this phenomenon further, vector isolates from pas-
sages 2, 6, and 12 of Ad2/CFTR-1 were analyzed at the mo-
lecular level. These analyses indicated that a deletion mutant
lacking sequences from within the CFTR cDNA had over-
grown both the parent vector and the RCA present at passage
3.
An Ad2 vector with rearranged pIX sequences. Despite the
advantage of reduction in RCA generation, deletion of pIX
sequences has two distinct disadvantages. First, the cloning
capacity of such vectors is limited because the vector genome

TABLE 1. Results of RCA assays

Resulta at dose of:
Ad vector Passage Titer (IU/ml)
1.25 3 109 IU 2.5 3 109 IU 2 3 1010 IU

Ad2/CFTR-1 1 2.2 3 109 P3, P6, P9, P12—Pass P3, P12—Pass P3-Fail
6 3.6 3 109 P12—Pass
Ad2/CFTR-2 1 7.2 3 109 P3, P6, P9, P12—Pass P3—Pass P3—Pass
6 2.2 3 109 P12—Fail with 4 plaques P12—Fail, 100% CPE
Ad2/CFTR-6 1 1.8 3 109 P3, P6, P9, P12—Pass P3, P12—Pass P3—Pass
7 3.0 3 109 P12—Fail with 20
plaques
Ad2/CFTR-7 1 3.4 3 107 P3, P6, P9, P1—Pass P3, P12—Pass P3, P12—Pass
7 1.9 3 108
a
Pass, Fail.
VOL. 70, 1996
FIG. 6. Thermal stabilities of Ad2/CFTR vectors. Samples of CsCl-purified
preparations of Ad2/CFTR-2, Ad2/CFTR-7, and Ad2/CFTR-8 were incubated in
microcentrifuge tubes in a water bath at 378C for the indicated times. The
samples were frozen at 2808C until the residual vector titers were determined.
Each point on the graph represents the average of duplicate titer determinations.
length must be less than 95% of the wild-type Ad2 genome
length (4, 6). For vectors carrying a large cDNA such as the
CFTR cDNA, at least a portion of the E3 region would also
have to be deleted so as not to exceed the vector size limit.
Second, the lack of pIX decreased the thermostability of Ad2/
CFTR-7 virions (Fig. 6), a phenomenon that has been reported
previously (4, 6). Finally, no additional information is available
on the biophysical properties of vectors lacking pIX. In partic-
ular, it was not known whether pIX-deleted virions would be
stable during nebulization, the probable method of adminis-
tration of Ad vector preparations for treatment of CF patients.
Therefore, it seemed that an alternative strategy would be
required to reduce recombination with 293 cell Ad5 sequences.
We reasoned that the reduction in recombination between
vector sequences and 293 cell Ad5 sequences accomplished by
deletion of pIX sequences could be accomplished as well by
movement of these sequences to a different location within the
Ad2 vector backbone. Retention of the pIX gene in this rear-
ranged vector would allow the packaging of Ad vector genomes
of full-length or greater size, which would in turn allow the
retention of the E3 genes and the large CFTR cDNA.
A new CFTR vector, Ad2/CFTR-8 (Fig. 5B), with relocated
pIX sequences was constructed in several steps as outlined in
Materials and Methods. Briefly, a plasmid containing the E4
ORF6 region was modified first by the addition of the SV40
early region polyadenylation signal sequence downstream from
the ORF6 sequence. This modification was carried out to pre-
vent the transcriptional readthrough that occurs in the original
ORF6 vector, Ad2/CFTR-2 (2). Next, the known sequences
comprising the minimal pIX promoter, amino acid coding re-
gion, and polyadenylation signal were amplified from Ad2
genomic DNA by PCR and inserted downstream from SV40
early region polyadenylation signal sequence and the E4 ORF6
cDNA. The pIX expression cassette in Ad2/CFTR-8 is tran-
scribed from right to left, in the same transcriptional orienta-
tion as the ORF6 gene. Modifications to the left end of the
genome were carried out such that Ad2 sequences between
REPLICATION-COMPETENT ADENOVIRUS STRUCTURE 8465
positions 546 and 4020 were deleted and replaced with the
CFTR cDNA and the SV40 early polyadenylation signal se-
quences. As a result of these alterations, the E1 promoter was
retained to control CFTR cDNA transcription and all of the
pIX promoter and amino acid coding sequences were deleted
to reduce the extent of homology with 293 cell Ad5 sequences.
The structure of the Ad2/CFTR-8 genome was confirmed by
restriction endonuclease mapping and nucleotide sequencing.
To be certain that adequate levels of pIX were produced from
the pIX gene in its rearranged configuration, the thermal sta-
bility of Ad2/CFTR-8 was tested and shown to be similar to
that of Ad2/CFTR-2 (Fig. 6).
To confirm that the relocation of the pIX sequences reduced
the incidence of recombination and RCA generation, a master
stock of Ad2/CFTR-8 and high-titer vector preparations made
from the master stock were tested for the presence of RCA. No
RCA were detected in a dose of 2.5 3 109 IU from the master
stock. More than 25 high-titer preparations of Ad2/CFTR-8
have been tested at doses ranging from 2.5 3 109 to 2.5 3 1010
IU, and only a single RCA plaque has been detected. The
structure of this RCA isolate as determined by restriction
endonuclease mapping is consistent with that of a chimera with
the right end derived from the Ad2/CFTR-8 genome and the
left end derived by recombination with 293 cell Ad5 E1 se-
quences.
DISCUSSION
Structure and origin of RCA. The primary goal of this study
was to provide greater understanding of the structure and
origin of RCA generated after growth of E1 replacement vec-
tors in 293 cells. By determining the molecular structure of
RCA isolates, we demonstrated that RCA are generated by
homologous recombination between vector and 293 cell Ad5
sequences. In four of five RCA isolates analyzed in detail,
recombination events both to the left and to the right of the
CFTR expression cassette led to the generation of RCA. In the
case of the RCA isolate from Ad2/CFTR-1, recombination
could have taken place within the first 150 nucleotides, where
the homology between Ad2 and Ad5 is complete and conse-
quently there is no way to establish the origin of sequences in
this region. RCA isolates derived from the different vectors
have unique structures, suggesting that recombination can take
place at many different points within the homologous vector-
derived Ad2 and 293 cell-derived Ad5 sequences.
By mapping the Ad5 sequences within 293 cells, we demon-
strated that the contiguous Ad5 sequences within the 293 cell
genome extended from near the left end of the Ad5 genome to
a position several hundred base pairs beyond the right-hand
deletion endpoint in simple E1 replacement Ad vectors. This
arrangement of sequences would be predicted to allow the
observed recombination events between vector and chromo-
somal DNA both to the left and to the right of the transgene.
Because dual recombination events predominate in the gener-
ation of RCA, it is evident that the terminal sequences from
the Ad5 genome derived from 293 cells are not usually incor-
porated into the RCA genome. Two hypotheses can be put
forward to explain these results: sequences from the extreme
terminus of the Ad5 genome could be deleted from the 293 cell
genome, and Ad5 terminal sequences could be present but
excised inefficiently from the 293 cell genome.
The result of such a recombination event is the formation of
an Ad2-Ad5 chimera that is capable of autonomous replication
due to the restoration of the Ad5 E1 region. No aberrant
recombinants were observed; thus, the biological properties
of such Ad2-Ad5 chimeras would be expected to be similar
8466 HEHIR ET AL.
to those of the parental virus vector backbone. In the case of
the Ad2/CFTR vectors described here (except Ad2/CFTR-1,
which retains the entire E4 region), each of the RCA isolates
has the modified E4 region derived from the parental vector
backbone. We have shown previously that the parental Ad2/
ORF6 virus is disabled compared with wild-type Ad2 (2); thus,
RCA arising from ORF6 vectors should be less pathogenic
than wild-type RCA. RCA arising from a vector backbone
bearing an E3 deletion, for example, the RCA isolate from
Ad2/CFTR-7 in this study and RCA arising from E3-deleted
vectors reported by Lochmüller et al. (13), would bear the E3
deletion as well. Results of preclinical studies in cotton rats
which are semipermissive for Ad replication have indicated
that deletion of E3 sequences increases the pathogenicity of
pulmonary adenovirus infection (7). Therefore, as the design
of Ad vectors continues to evolve, it is important to consider
that genes in wild-type or mutant forms that may be incorpo-
rated into vector genomes could ultimately become incorpo-
rated into RCA. These considerations also highlight the im-
portance of an effective and sensitive assay for RCA in vector
preparations that is designed to detect the vector backbone in
clinical use.
Rate of RCA formation. The blind passage experiment was
carried out in an attempt to compare the relative rates of RCA
formation from the different vectors. Each of the vectors was
purified by limiting dilution at the outset of the experiment,
passaged at a low MOI (;5), and assayed for RCA at passages
3, 6, 9, and 12. Under these conditions, no RCA were detected
in any vector at a dose of 1.25 3 108 IU. As expected from our
experience with our first-generation Ad2 vector, Ad2/CFTR-1,
RCA were detected at an earlier passage than the other vec-
tors. This is probably a consequence of the fact that Ad2/
CFTR-1 has poor growth properties and is readily overgrown.
Each of the other vectors tested grows better than Ad2/
CFTR-1, and additional passages or higher test doses were
required to reveal the presence of RCA.
Lochmüller et al. (13) have reported the generation of RCA
from Ad vectors after continuous passage, with the first indi-
cations of RCA genomes at passage 12. Estimates of the con-
tamination of the viral vector population with RCA were from
1 to 20%, depending on the assay used. The least sensitive
assay used in the current study can detect 5 to 10 RCA ge-
nomes in a test dose of 1.25 3 108 IU; i.e., it is approximately
100-fold more sensitive than the most sensitive assay used by
Lochmüller et al. (13). With the more sensitive assay, it is likely
that Lochmüller et al. (13) would have detected the presence
of RCA at an earlier passage. Thus, the two studies are in
general agreement regarding the rate at which RCA are gen-
erated by passaging of conventional E1 replacement vectors on
293 cells.
Vector backbone changes to reduce RCA formation. On the
basis of the analysis of the Ad5 sequences in the 293 cell
genome and of RCA genomes generated from different Ad2/
CFTR vectors, we reasoned that it would be feasible to reduce
the frequency of recombination leading to RCA by altering the
structure of the vector backbone. Modification of the extreme
left end of the genome appeared problematic because of the
presence of packaging signals required in cis for efficient vector
growth. Therefore, we considered whether sequences on the
right side of the transgene could be deleted to reduce the
likelihood of recombination. It has been shown that pIX func-
tion is not required for virus growth or for packaging of viral
genomes less than 95% of wild-type genome length. Our anal-
ysis of RCA genomes revealed that the pIX gene accounted for
50 to 60% (depending on the vector) of the homology between
vector and 293 cell Ad5 sequences on the right side of the
J. VIROL.
transgene. Thus, deletion of the pIX gene would have been
expected to reduce the frequency of recombination that leads
to RCA formation. The vector Ad2/CFTR-7 was constructed
to test this hypothesis. As demonstrated by the results pre-
sented in Table 1, deletion of pIX sequences had the predicted
effect on reduction of RCA generation. No RCA were de-
tected even when this vector was carried through 12 blind
passages.
The Ad2/CFTR-8 vector, with its novel arrangement of the
pIX gene, was constructed to take advantage of the reduction
in RCA generation exhibited by Ad2/CFTR-7 while retaining
the capability for packaging full-length vector genomes into
virions that have normal thermostability. As we have shown,
the arrangement of sequences within this vector reduces the
occurrence of RCA in high-titer vector preparations to approx-
imately the same extent as the complete deletion of the pIX
gene. The structure of the single RCA isolate detected suggests
that recombination took place between Ad2 nucleotide posi-
tions 4020 and ;4300, thus incorporating the entire E1-pIX
region from 293 cell Ad5 sequences. In this isolate, the Ad2
pIX gene is retained at the right-hand end of the genome (data
not shown). Thus, it is clear that deletion or rearrangement of
pIX sequences will reduce significantly but not eliminate the
possibility for RCA generation by recombination with 293 cell
Ad5 sequences. We also recognize the potential for other re-
combination events between the Ad2/CFTR-8 vector and 293
cell Ad5 sequences, events that do not lead to the formation of
stable viral genomes. For example, it is possible for 293 cell
Ad5 pIX sequences to pair with the Ad2 pIX sequences in
their novel location and for recombination to occur. The full
range of recombinant structures resulting from such recombi-
nation events are difficult to predict. However, because of the
inverted orientation of the pIX gene within Ad2/CFTR-8 and
its positioning relative to the ORF6 expression unit, most if not
all recombinants will be replication defective due to deletion of
the E4 region and the right terminus of the genome.
It should be noted that relocation of the pIX gene to a
different position within the vector genome is not the only
strategy that might be effective in reducing RCA generation
while providing pIX for maximal vector stability. Cell lines with
the pIX gene transduced into the 293 cell which provide suf-
ficient levels of pIX for vector packaging have been developed
(3, 12). As an alternative to the 293 cell, an A549-based pro-
ducer cell line in which there are no sequences in common
between the integrated Ad E1 genomic segment and the Ad
vector genome, thus precluding generation of RCA by homol-
ogous recombination, has been developed (9).
Genetic instability of virus vectors. To date, the primary
focus on genetic variants within Ad-based gene therapy vector
populations has been on RCA. This focus stems from concerns
about the disease-causing potential of essentially wild-type Ad,
which is fairly mild with serotypes Ad2 and Ad5. We have
shown here that the basic vector structure can be altered to
eliminate practically the generation of RCA. The results pre-
sented here point out the need to be aware of other types of
variants and to consider their impact on preparations of Ad
gene therapy vectors. As ever-larger regions of genes and as-
sociated regulatory elements are cloned into Ad vectors, it is
predictable that more complex and perhaps more frequent
sequence rearrangements will occur with the potential for af-
fecting expression of the therapeutic gene. It is therefore cru-
cial to establish sensitive activity assays for the therapeutic
gene product to ensure that the impact of development of
subpopulations of recombinants is kept to a minimum.
VOL. 70, 1996
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