Table Of ContentCurrent Topics In
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Adeno-Associated Virus (AAV)
Vectors in Gene Therapy
Edited by K.I. Berns and C. Giraud
With 38 Figures
, Springer
Professor KENNETH I. BERNS, M.D., Ph.D.
CATHERINE GIRAUD, Ph.D.
Cornell University Medical College
Hearst Microbiology Research Center
Department of Microbiology
1300 York Avenue, Box 62
New York, NY 10021
USA
Cover illustration: The inverted terminal repeat of the adeno
associated virus genome is shown in the folded conformation
which maximizes base pairing, The inverted terminal repeat
plays a significant role in the regulation of viral gene expression,
DNA replication, and integration into and excision from the
integration site in the human genome. The red letters indicate
the binding site of the viral regulatory protein "rep 68/78."
"trs" indicates the site nicked by the rep 68/78 protein.
Cover design: Design & Production, Heidelberg
ISSN 0070-217X
ISBN-13: 978-3-642-80209-6 e-ISBN-13: 978-3-642-80207-2
001: 10.1007/978-3-642-80207-2
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© Springer-Verlag Berlin Heidelberg 1996
Softcover reprint of the hardcover 1 st edition 1996
Library of Congress Catalog Card Number 15 -12910
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Preface
Human gene therapy holds great promise for the cure of many
genetic diseases. In order to achieve such a cure there are two
requirements. First, the affected gene must be cloned, its se
quence determined and its regulation adequately characterized.
Second, a suitable vector for the delivery of a good copy of the
affected gene must be available. For a vector to be of use
several attributes are highly desirable: these include ability to
carry the intact gene (although this may be either the genomic
or the cDNA form) in a stable form, ability to introduce the gene
into the desired cell type, ability to express the introduced gene
in an appropriately regulated manner for an extended period of
time, and a lack of toxicity for the recipient. Also of concern is
the frequency of cell transformation and, in some cases, the
ability to introduce the gene into nondividing stem cells. Sev
eral animal viruses have been tested as potential vectors, but
none has proven to have all the desired properties described
above. For example, retroviruses are difficult to propagate in
sufficient titers, do not integrate into nondividing cells, and are
of concern because of their oncogenic properties in some
hosts and because they integrate at many sites in the genome
and, thus, are potentially insertional mutagens. Additionally,
genes introduced by retroviral vectors are frequently expressed
for relatively short periods of time. A second virus used as a
vector in model systems has been adenovirus (Ad). The major
deficit is that Ad does not integrate and functions as a transient
vector, necessitating repeated administration with the possi
bility of a toxic immune response. A third virus of interest as a
possible vector is adeno-associated virus (MV), a small DNA
virus which has many of the properties desired in a vector. This
volume is devoted to descriptions of the manner in which MV
has been used as a vector for gene therapy in cell culture and in
model animal systems. The initial chapter is a general overview
of the biology and molecular biology of M V with an emphasis
on those properties directly pertinent to use as a vector. Also
included are discussions of some of the questions that remain
VI Preface
to be answered before the possibility of clinical application.
Subsequent chapters describe the preparation of AAV vectors,
including attempts to develop packaging cell lines. The re
maining chapters describe the construction and use in vitro of
AAV virion vectors for the potential treatment of diseases of
the hematopoietic system, cystic fibrosis, and AIDS. Two
fundamental questions remain to be resolved. The first is
practical: how to produce vectors in sufficient quantity for
clinical use. The second is both theoretical and practical: should
AAV vectors be designed to integrate at a specific site in the
human genome. The reader should consider both of these is
sues as the chapters on the use of AAV as a vector are read.
July 1996 KENNETH I. BERNS
List of Contents
K.I. BERNS and C. GIRAUD
Biology of Adeno-associated Virus
J.A. CHIORINI, S.M. WIENER, L. YANG, R.H. SMITH,
B. SAFER, N.P. KILCOIN, Y. LIU, E. URCELAY,
and R.M. KOTIN
The Roles of AAV Rep Proteins in Gene Expression
and Targeted Integration ...................... 25
J.P. TREMPE
Packaging Systems
for Adeno-associated Virus Vectors 35
o
J.S. LEBKOWSKI, T.B. KARMA, and R. PHILIP
The Challenges of Recombinant Adeno-associated
Virus Manufacturing: Alternative Use
of Adeno-associated Virus Plasmid/Liposome
Complexes for Gene Therapy Applications ......... 51
S. CHATTERJEE and K.K. WONG JR.
Adeno-associated Virus Vectors for Gene Therapy
of the Hematopoietic System. . . . . . . . . . . . . . . . . . . 61
D.M. MCCARTY and R.J. SAMULSKI
Adeno-associated Virus Vectors
for Gene Transfer into Erythroid Cells 75
A. SRIVASTAVA, X.-S. WANG, S. PONNAZHAGAN,
S.Z. ZHOU, and M.C. YODER
Adeno-associated Virus 2-Mediated Transduction
and Erythroid Lineage-Specific Expression
in Human Hematopoietic Progenitor Cells . . . . . . . . . . 93
B.J. CARTER and T.R. FLOTTE
Development of Adeno-associated Virus Vectors
for Gene Therapy of Cystic Fibrosis .............. 119
K.K. WONG JR. and S. CHATTERJEE
Adeno-associated Virus-Based Vectors As Antivirals .. 145
Subject Index .............................. 171
List of Contributors
(Their addresses can be found at the beginning of their
respective chapters.)
BERNS, K.1. 1 PONNAZHAGAN, S. 93
CARTER, B.J. 119 SAFER, B. 25
CHATIERJEE, S. 61, 145 SAMULSKI, R.J. 75
CHIORINI, J .A. 25 SMITH, R.H. 25
FLOTIE, T.R. 119 SRIVASTAVA, A. 93
GIRAUD, C. 1 TREMPE, J.P. 35
KILCOIN, N.P. 25 URCELAY, E. 25
KOTIN, R.M. 25 WANG, X.-S. 93
LEBKOWSKI, J.S. 51 WIENER, S.M. 25
LIU, Y. 25 WONG, K.K., JR. 61, 145
MCCARlY, D.M. 75 YANG, L. 25
o
KARMA, T.B. 51 YODER, M.C. 93
PHILIP, R. 51 ZHOU, S.Z. 93
Biology of Adeno-associated Virus
K.I. BERNS and C. GIRAUD
1 Adeno-associated Virus Life Cycle .
2 Genetics .. 4
3 Latent Infection 6
4 Rescue and Replicative Infection. 12
5 Adeno-associated Virus Vectors for Gene Therapy 14
References ... 18
1 Adena-associated Virus Life Cycle
Adeno-associated Virus (AAV) is classified as a member of the family Parvovi
ridae (SIEGL et al. 1985; BERNS 1990a). Members are small, nonenveloped, ico
sahedral viruses (diameter ca. 20-26 nm) with linear, single-stranded DNA
genomes of 4.7-6 kb. Parvoviridae have been isolated from many species
ranging from insects to humans. AAV is assigned to the genus Oependovirus, so
named because the virus was discovered as a contaminant in purified adeno
virus (Ad) stocks and in most instances does not productively infect cells in
culture unless there is a coinfection by an unrelated helper virus, which is most
commonly Ad, but also can be any type of herpesvirus (ATCHINSON et al. 1965;
HOGGAN et al. 1966; BULLER et al. 1981). Various serotypes have been isolated
from birds and many mammalian species, including humans. About 90% of U.S.
adults are seropositive, but in no case has the virus been implicated as the
etiological agent for a human disease or as the cause of disease in any other
species. Because of the requirement for a helper coinfection for productive
infection in cell culture, AAV was long considered to be a defective virus. De
tailed studies described below have demonstrated that the virus is not defective,
but rather preferentially establishes a latent infection in a healthy cell and is only
induced to undergo productive vegetative multiplication when the host cell is
stressed.
Department of Microbiology, Hearst Microbiology Research Center, Cornell University Medical
College, 1300 York Avenue, New York, NY 10021, USA
2 K.I. Berns and C. Giraud
•AAV
\
Integrot1on In
chromosome 1 9
~-I
AAVDNA
Rescue
H
H
/RePllcellon
• •
••••
Fig. 1. Adeno-associated virus (AAV) life cycle. Under nonpermissive conditions AAV integrates into
the q arm of human chromosome 19 where it remains silent until challenged by a helper virus, e.g.,
adenovirus. This leads to rescue of the integrated virus from the chromosome and induction of the
lytic cycle. Under permissive conditions, i.e., in the presence of a helper virus, such as adenovirus,
AA V replicates, resulting in host cell lysis
