Advances in Genetics Volume 55

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Volume 55

Advances in Genetics

Advances in Genetics, Volume 55

Serial Editors

Jeffery C. Hall Waltham, Massachusetts

Jay C. Dunlap Hanover, New Hampshire

Theodore Friedmann La Jolla, California

Veronica van Heyningen Edinburgh, United Kingdom

Volume 55

Advances in Genetics Edited by

Jeffrey C. Hall Department of Biology Brandeis University Waltham, Massachusetts

Jay C. Dunlap Department of Biochemistry Dartmouth Medical School Hanover, New Hampshire

Theodore Friedmann Center for Molecular Genetics University of California at San Diego School of Medicine La Jolla, San Diego, California

Veronica van Heyningen MRC Human Genetics Unit Western General Hospital Edinburgh, United Kingdom

AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO Academic Press is an imprint of Elsevier

Elsevier Academic Press 525 B Street, Suite 1900, San Diego, California 92101-4495, USA 84 Theobald’s Road, London WC1X 8RR, UK

This book is printed on acid-free paper. Copyright ß 2005, Elsevier Inc. All Rights Reserved.

No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (www.copyright.com), for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-2005 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 0065-2660/2005 $35.00 Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone: (þ44) 1865 843830, fax: (þ44) 1865 853333, E-mail: [email protected]. You may also complete your request on-line via the Elsevier homepage (http://elsevier.com), by selecting ‘‘Customer Support’’ and then ‘‘Obtaining Permissions.’’ For all information on all Elsevier Academic Press publications visit our Web site at www.books.elsevier.com ISBN-13: 978-0-12-017655-7 ISBN-10: 0-12-017655-6 PRINTED IN THE UNITED STATES OF AMERICA 05 06 07 08 09 9 8 7 6 5 4 3 2 1

Contents Contributors

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1 Site-Specific DNA Recombinases as Instruments for Genomic Surgery 1 Aram Akopian and W. Marshall Stark I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII.

Introduction 2 Inadequacy of Current Methods 2 Site-Specific Recombination 4 Mechanisms of Site-Specific Recombination 5 Transposition 8 Applied Site-Specific Recombination and Transposition Systems 9 Changing Recombination Site Specificity 9 Mutagenesis-Selection Strategies 10 Structure-Based Strategies: Z-Resolvases 12 Targeting Transposition to Specific Sequences 15 General Considerations in Applications of Site-Specific Recombinases and Transposases 16 Prospects and Conclusions 18 References 19

2 Human Clinical Trials of Plasmid 25 DNA Vaccines Margaret A. Liu and Jeffrey B. Ulmer I. II. III. IV. V.

Background 26 Development of DNA Vaccines for Clinical Trials Human Clinical Trials 31 Limitations of DNA Vaccine Potency 33 Prospects and Conclusion 35 References 35

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3 Genetic and Environmental Influences on Antisocial Behaviors: Evidence from Behavioral–Genetic Research 41 Terrie E. Moffitt I. Appraising the Quantitative Behavioral–Genetic Evidence Base 44 II. Estimating the Relative Influences of Genes and Environments 58 III. Do the Omnibus Estimates of Genetic and Environmental Influences Always Apply, or do These Influences Vary Under Different Conditions? 66 IV. Testing Developmental Theory of Antisocial Behavior 71 V. Testing Hypotheses About Environmental Causation 74 VI. Testing the Hypothesis of Interaction Between Genes and Environments 80 VII. The Way Forward 88 References 91

4 Genetics of Graviperception in Animals

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Kathleen M. Beckingham, Michael J. Texada, Dean A. Baker, Ravi Munjaal, and J. Douglas Armstrong I. Introduction 106 II. Vertebrate Genetic Models (M. musculus and D. rerio) 107 III. The Arthropod Genetic Model (D. melanogaster) IV. The Nematode Model System (C. elegans) 135 V. Conclusion 137 References 138

5 Retroviral DNA Integration—Mechanism and Consequences 147 Mary K. Lewinski and Frederic D. Bushman I. Introduction 148 II. Retroviral Life Cycle 148 III. Mechanism of Integration 149

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Contents

IV. V. VI. VII. VIII.

Integrase Structure 153 Composition of Integrase Complexes In Vivo 159 Retroviral Integration Targeting 162 Consequences of Integration into Host Chromosomes Conclusions 169 References 170

Index

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Contributors Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Aram Akopian (1) Institute of Biomedical & Life Sciences, University of Glasgow, Glasgow G11 6NU, United Kingdom J. Douglas Armstrong (105) School of Informatics, University of Edinburgh, Edinburgh EH8 9LE, United Kingdom Dean A. Baker (105) School of Informatics, University of Edinburgh, Edinburgh EH8 9LE, United Kingdom Kathleen M. Beckingham (105) Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005 Frederic D. Bushman (147) Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 Mary K. Lewinski (147) Infectious Disease Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92186 Margaret A. Liu (25) Transgene S.A., 11, rue de Molsheim, 67082 Strasbourg Cedex, France Terrie E. Moffitt (41) Institute of Psychiatry, King’s College London, SE5 8AF, United Kingdom; University of Wisconsin, Madison, Wisconsin 53706 Ravi Munjaal (105) Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005 W. Marshall Stark (1) Institute of Biomedical & Life Sciences, University of Glasgow, Glasgow G11 6NU, United Kingdom Michael J. Texada (105) Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005 Jeffrey B. Ulmer (25) Chiron Corporation, Emeryville, California 94608

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Site‐Specific DNA Recombinases as Instruments for Genomic Surgery Aram Akopian and W. Marshall Stark Institute of Biomedical & Life Sciences University of Glasgow, Glasgow G11 6NU United Kingdom

I. II. III. IV. V. VI. VII. VIII. IX. X. XI.

XII.

Introduction Inadequacy of Current Methods Site‐Specific Recombination Mechanisms of Site‐Specific Recombination Transposition Applied Site‐Specific Recombination and Transposition Systems Changing Recombination Site Specificity Mutagenesis‐Selection Strategies Structure‐Based Strategies: Z‐Resolvases Targeting Transposition to Specific Sequences General Considerations in Applications of Site‐Specific Recombinases and Transposases A. Recombinase and transgene “delivery” B. Chromatin C. Reversibility D. Nonspecific reactions Prospects and Conclusions References

Advances in Genetics, Vol. 55 Copyright 2005, Elsevier Inc. All rights reserved.

0065-2660/05 $35.00 DOI: 10.1016/S0065-2660(05)55001-6

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ABSTRACT Site‐specific DNA recombinases can “cut and paste” DNA. For example, they can promote excision of specific DNA segments or insertion of new DNA segments in specific places. However, natural recombinases act only at their cognate recombination sites, so current applications are limited to genetically modified organisms in which these sites have been introduced into the genome. Transposases also catalyze DNA rearrangements; they promote insertion of specific DNA sequences but at nonspecific locations. Applicability of site‐ specific recombinases and transposases in experimental genetics, biotechnology, and gene therapy would be much wider if they could be reengineered so as to act specifically at chosen sequences within an organism’s natural genome. This review will discuss progress towards the creation of such “designer” recombinases. ß 2005, Elsevier Inc.

I. INTRODUCTION As numerous genomes are sequenced and our understanding of genome function grows, the possibilities for changing genomic DNA sequence to our benefit become more apparent. We could add new useful bits of DNA in specific places and delete or change defective or undesirable bits. This “genomic surgery” could be applied to the treatment of disease as well as in biotechnology and experimental genetics. The development of realistic methods for genomic surgery will bring many opportunities for improving our health and well being, along with dangers and ethical problems that will be faced by science and society. In this review, we will discuss advances in the still‐primitive methods that are under consideration, focusing on the attempts to adapt natural enzymes that “cut and paste” DNA—recombinases and transposases—so that their activity can be directed to chosen sequences.

II. INADEQUACY OF CURRENT METHODS For many years, it has been possible to make artificial alterations to an organism’s genetic material. The alteration may be temporary (e.g., when foreign nonreplicating DNA is introduced into a cell or when gene expression is altered by RNAi) or permanent (e.g., when transgenic DNA integrates into a chromosome). Integration of a foreign DNA segment can be targeted to a specific genomic locus by methods based on homologous recombination (HR; see in later section). Why then do we need new methods to target DNA manipulation to particular genomic sites?

1. Site-Specific DNA Recombinases as Instruments for Genomic Surgery

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The currently available methods for directing DNA modification to specific target sequences lack the versatility, efficiency, and specificity, which will be needed if genomic surgery is to take off (Yanez and Porter, 1998). For example, consider a scenario in which a new functional gene is to be integrated into a mammalian genome. Without targeting, a transfected DNA fragment will generally integrate inefficiently and at essentially random positions (Lacy et al., 1983; Smith, 2001). Transgene expression might therefore be subject to locus‐ specific downregulation by epigenetic silencing processes (Pannell and Ellis, 2001) or the integrated DNA might affect expression of nearby genes, thus potentially causing cell malfunction or oncogenesis (Palmiter and Brinster, 1986). Ideally, the transgene should be integrated at a locus where it will be expressed properly, and it will not interfere with other functions of the genome. Use of HR is the only current means of targeting integration to any chosen genomic locus. By embedding the gene to be transfected in the middle of a few kilobase‐pairs (kbp) of target site sequence, the cell’s HR machinery can be co‐ opted to promote integration at that site. This approach has been successfully applied as an experimental tool in some organisms for integration of new genes or to “knock out” resident genes (e.g., Colosimo et al., 2000; Vasquez et al., 2001). Unfortunately, integration by HR is inefficient, and specificity for the chosen target locus is low. Normally, the great majority of cells following transfection have not integrated the transfected DNA, and the transgene lands at the desired target site in only a small fraction of the remainder (Smith, 2001; Vasquez et al., 2001). Homologous recombination is therefore of practical use only if one can select the correctly modified cells and, if necessary, induce them to participate in embryogenesis. The options for improvement of HR‐based methods are limited because HR is crucial for the maintenance of the genome; tampering with its natural components will possibly have serious biological consequences and would probably only be feasible in experimental systems. A further technical limitation of HR is that the transgene must be flanked by long sequences homologous to the target. This can restrict the size of genes to be introduced with viral vectors, which can only carry a limited length of foreign sequence (Monahan and Samulski, 2000; Yanez and Porter, 1998). Methods for enhancing the efficiency of HR at chosen sites have been developed, based on the introduction of factors with sequence recognition specificity (DNA‐binding proteins or oligonucleotides) (Uil et al., 2003). However, it seems that, for more advanced genomic surgery, alternatives to HR will be required. In later sections, we consider whether site‐specific recombinases and transposases may be useful “surgical instruments.” Other strategies for alteration of specific genomic sequences are also being investigated (Belfort et al., 2002; Collins et al., 2003; Epinat et al., 2003; Guo et al., 2000; Lambowitz and Zimmerly, 2004; Monahan and Samulski, 2000; Portlock and Calos, 2003; Uil et al., 2003).

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III. SITE‐SPECIFIC RECOMBINATION Site‐specific recombinases rearrange DNA sequences by catalyzing cleavage and rejoining of DNA strands at specific short DNA sequences (sites) to which they bind. The outcome of the reaction can be excision of a DNA sequence segment bounded by two sites, or the reverse of this process, integration, or inversion of the orientation of a segment of DNA bounded by two sites (Fig. 1.1). An attractive feature of these systems as potential tools for genomic surgery is that they can be highly specific, efficient, and fast (Nash, 1996). Microbial site‐specific recombination systems have been widely exploited in mammals and other higher eukaryotes for experimental research or projected gene therapy/biotechnology applications (see Section VI). However, their use is currently limited to situations where one or more of the sites recognized by the recombinase have already been introduced into the genome by other methods. It would be a major advance if we could engineer recombinases to act with high efficiency and specificity at sequences that are already there. The best‐characterized site‐specific recombination systems come from bacteria and yeasts. Most of the systems can be assigned to one of two large families, according to the type of recombinase that is used. These two families, the tyrosine recombinases and the serine recombinases, are so‐called because the conserved nucleophilic amino acid residue that attacks and becomes linked to the DNA during strand exchange is either a tyrosine or a serine. The well‐ known enzymes Cre, FLP, and integrase are tyrosine recombinases; examples from the serine recombinase family are resolvase and C31 integrase (Nash, 1996). The two families are unrelated to each other, having different protein structures and reaction mechanisms.

Figure 1.1. Substrates and products of site‐specific recombination reactions. The diagrams show how the relationship of two recombining sites (small black and white arrows) determines the type of recombination product formed. On the left, excision of DNA between directly repeated sites and the reverse reaction, integration; on the right, inversion of the DNA segment between sites in inverted repeat.


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Natural site‐specific recombination systems have evolved to carry out specific “programmed” genetic rearrangements in their host organisms. Therefore, their activity is usually tightly regulated, and the type of rearrangement they bring about (excision, inversion, or integration) is usually specified. These constraints can cause problems for those who wish to subvert the recombinases to become tools for genetic manipulation.

IV. MECHANISMS OF SITE‐SPECIFIC RECOMBINATION The two DNA sites that are to recombine (typically a few tens of base‐pains(bp) long; Fig. 1.2B) must first be recognized by the recombinase protein. The protein‐bound sites must then be brought together (synapsed) prior to strand exchange, which involves cutting and rejoining of the DNA strands. During strand exchange, the recombinase transiently becomes covalently linked to a phosphate of the DNA backbone via the hydroxyl group of the nucleophilic tyrosine or serine residue (Hallet and Sherratt, 1997; Nash, 1996). The strands are broken at fixed points in the site sequence. Tyrosine recombinases exchange strands one pair at a time, and thus the reaction proceeds via an intermediate which is analogous to the Holliday junction of HR (Holliday, 1964). In contrast, serine recombinases make intermediates in which all four DNA strands of the two recombination sites are broken. These mechanisms are summarized in Fig. 1.2A. The recombination site generally has an asymmetric sequence and thus a “left” and a “right” end; each left end is joined to the right end of the partner site, so as to reconstitute similar sites in the recombination products. This polarity is often assured by an asymmetric “overlap” sequence between the staggered points of top and bottom strand exchange, so that recombination of misaligned sites would produce mismatched base pairs in the recombinants (Fig. 1.2B). The connectivity of the two recombining sites in the substrate DNA therefore defines the type of product formed: inversion, if the sites are oriented in opposite directions to each other; excision, if they are directly repeated (“head to tail”); and integration, if they are on separate molecules (Fig. 1.1). Some recombinases will act on pairs of sites in any relationship (e.g., Cre and Flp), whereas others are specific for one type of relationship (e.g., resolvase, which only catalyzes excision). Our understanding of the mechanisms of site‐specific recombination has been greatly advanced by a number of crystal structures showing recombinases bound to their cognate sites, and intermediates in the strand exchange process itself (Chen et al., 2000; Grindley, 2002; Guo et al., 1999; Van Duyne, 2002; Yang and Steitz, 1995). Structures of Cre (Fig. 1.3) and Flp show a recombinase tetramer synapsing two recombination sites. Each subunit of these tyrosine recombinases wraps around the DNA, making many contacts with it.

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Figure 1.2. (A) Mechanisms of DNA strand exchange by serine recombinases and tyrosine recombinases. The pairs of thick or thin lines represent double‐stranded DNA. The small arrowheads mark the ends of a “site” of a few tens of basepairs (Fig. 1.2B), which is recognized specifically by the recombinase enzyme (not shown). The staggered lines show where the strands are broken at the centres of the sites. Note that the sites are aligned in parallel in the upper row (serine recombinases), but in antiparallel in the lower row (tyrosine recombinases), reflecting the proposed structures of reaction intermediates. See text for further details. (B) Recombination sites. The sequences of two simple recombination sites are shown: loxP, the site for Cre recombinase, and attB, one of the two nonidentical sites for C31 integrase. The loxP site has twofold (palindrome) symmetry, indicated by the long arrows, except in the central 8 bp; in contrast, C31 attB is quite asymmetric. The short black arrows indicate the positions of breakage and rejoining of the DNA strands. The asymmetric ‘overlap’ sequence between the two arrows is important in specifying that left and right half‐sites are joined in the recombinant products. The overlap is typically 2 bp for serine recombinase sites, as in C31 attB, and 5–8 bp for tyrosine recombinase sites (6 bp in loxP).

The basis of sequence‐specific binding by these proteins is still not clear. A structure of the serine recombinase resolvase (Fig. 1.3) shows a recombinase dimer bound to a single site. A small C‐terminal domain of each resolvase subunit, thought to be responsible for much of the sequence specificity, binds in the major groove of the DNA. The larger N‐terminal domains, which contain the active site and make intersubunit interactions, contact the DNA in the minor groove on the opposite side of the double helix from the C‐terminal domains.


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Figure 1.3. Crystal structures of site‐specific recombinases bound to DNA. (A) A dimer of the serine recombinase resolvase, bound to ‘site I’, the part of its recombination site where strand exchange occurs (Yang and Steitz, 1995). (B) A tetramer of the tyrosine recombinase Cre in a synapse with two loxP sites (Guo et al., 1999). The DNA (cream and blue) is in spacefill representation, and the protein (green and yellow) is in a ribbon representation. The two loxP sites in B are in approximately antiparallel alignment (see Fig. 1.1). The pictures were created with the program PYMOL. (See Color Insert.)

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V. TRANSPOSITION Transposition has much in common with site‐specific recombination. A semiautonomous DNA element called a transposon moves from one locus to another in a reaction catalyzed by a transposase enzyme. The ends of the transposon must be recognized and acted upon by the transposase site‐specifically, but the transposon DNA is then inserted at new positions, which are generally not specific (Fig. 1.4). The mechanisms of transposition are diverse (Craig, 2002a) and can even involve RNA intermediates (the process being then called retrotransposition). DNA transposases (those that catalyze direct transfer of DNA from one locus to another) belong to several different structural groups. Some are related to the serine and tyrosine site‐specific recombinases introduced in Section IV. The best characterized are the so‐called “DDE” transposases, which are very widespread; transposons that encode them are found in bacteria, archaea, and eukarya. Like site‐specific recombinases, transposases have already attracted considerable interest as potential tools for gene therapy and biotechnology (Boeke, 2002). They share with site‐specific recombinases the ability to promote highly efficient, specific, and fast reactions. By their nature, transposases are best adapted to promote integration of a DNA segment, flanked by short “ends” from their cognate transposon, into target DNA. The transposase breaks either one or both strands at each end and then joins the broken ends to the target DNA. The complete process normally involves subsequent “tidying‐up” operations, which require host enzymes (Craig, 2002a). Usually, the transposase has little or no selectivity for specific target sequences, so transposon insertions can occur

Figure 1.4. Transposition. In the simplest case, as shown here, a DNA transposon (stippled rectangle) is excised from the DNA by its transposase enzyme acting at specific end sequences (arrowheads), and then inserted into a random site in target DNA (thicker lines).


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anywhere in the genome; for exceptions that do have strongly preferred target sites or insert in specific regions of the genome, see Bushman, 2003; Craig, 2002b. The long terminal repeat (LTR) retrotransposons and LTR retroviruses reverse‐transcribe their RNA genome into DNA before integrating it into the host genome. Their integrase enzymes belong to the DDE transposase family, and the mechanism of integration is similar to that of typical DNA transposons with DDE transposases (Craig, 2002a). Integration by the non‐LTR retrotransposons involves different types of enzymes and direct interaction of RNA molecules with the target DNA. Non‐LTR retrotransposition systems may also have potential for targeted gene integration (Eickbush, 2002). In Section X, we will briefly review attempts to target insertion mediated by DDE transposases/retroviral integrases.

VI. APPLIED SITE‐SPECIFIC RECOMBINATION AND TRANSPOSITION SYSTEMS Recombinases have become very popular tools for manipulating DNA in vitro and in vivo (Boeke, 2002; Branda and Dymecki, 2004; Gorman and Bullock, 2000; Groth and Calos, 2003; Kilby et al., 1993; Kolb, 2002; Nagy, 2000). The most frequently used enzymes have been Cre recombinase from bacteriophage P1 and Flp recombinase from Saccharomyces cerevisiae. The 38‐kDa Cre protein promotes recombination between two 34‐bp loxP sites, and the 43‐kDa Flp recombinase acts on 34‐bp FRT sites. Advantages of these systems are their short DNA recombination sites, enzyme stability in vivo, and the robustness of their activity even when acting upon chromatin‐associated DNA (Jayaram et al., 2002; Sauer, 2002). Numerous other recombinases have been used in biotechnology or investigated as possible biotechnology tools (Boeke, 2002). Similarly, several DNA transposition systems have become popular for sequencing, mutagenesis, and transgene integration purposes (Boeke, 2002); and retroviral integration is being widely investigated for possible uses in gene therapy (see Sections X and XI).

VII. CHANGING RECOMBINATION SITE SPECIFICITY Hundreds of different natural site‐specific recombination systems have been identified, so there is a large natural “library” of sites with different sequences, acted upon by known recombinases. However, it is still very unlikely that any of these sequences will be found by chance in a useful place in a genome of interest, as most natural recombination sites are at least about 25 bp long. Furthermore,

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characterization of a new system to the stage where it could be used as a research tool requires a considerable amount of work. A more practical approach is to mutate or redesign a well‐characterized recombinase so that it recognizes a new sequence. Changing the sequence recognition properties of site‐specific recombinases has been a subject of investigation for many years; initially the aims were to provide insight on structure and mechanism and to create useful experimental tools. Recognition by serine recombinases related to resolvase has been altered by point mutation (Grindley, 1993) or by replacing part or all of the entire DNA‐binding domain with the equivalent sequence from a related protein (Ackroyd et al., 1990; Avila et al., 1990; Schneider et al., 2000). Similar mutation and domain‐swap approaches were used to change the specificities of tyrosine recombinases (Dorgai et al., 1995; Shaikh and Sadowski, 2000; Yagil et al., 1995). Recently, researchers have begun to develop more systematic strategies for altering recombinase site recognition, with the long‐term goal of creating more useful and adaptable systems for genetic manipulation in vivo. Two types of strategy are discussed in Sections IV and V. As hinted previously, it would be a great advance if integration could be targeted efficiently and specifically to even one good natural site in a genome of interest (e.g., human), where gene expression is optimal and there would be no adverse effects. One approach is therefore to choose a natural sequence with some similarity to the site for a useful recombinase, often one that has been shown to act as a “pseudosite” (where the recombinase occasionally catalyzes recombination). One can then attempt to produce variants of the recombinase that act there efficiently and specifically by multiple rounds of mutagenesis and selection. A more radical approach is the attempt to design a recombinase whose sequence specificity can be altered at will by changing a “DNA recognition module.”

VIII. MUTAGENESIS‐SELECTION STRATEGIES C31 Int, a serine recombinase, mediates integration by recombination of phage attP (39 bp) and bacterial genomic attB (34 bp) sites (Groth and Calos, 2003). Integration is irreversible in the reconstituted system lacking factors encoded by the phage C31 or its Streptomyces host. The relatively short sites and irreversible reaction make the C31 Int system a very attractive candidate for applications. There is no high‐resolution structural information as yet on C31 Int or any of its close relatives, but current biochemical evidence suggests that DNA recognition involves multiple domains within these large proteins. Systematic redesign of sequence specificity by “protein engineering” is therefore not an option at present. Although there are high‐resolution structures of the tyrosine recombinases Cre and Flp bound to their cognate sites, a protein‐engineering


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approach is still problematic for them because it is difficult to distinguish residues involved in DNA recognition from those involved in catalysis (Section IV). The mutagenesis‐selection approach for altering site recognition, which does not depend on a deep understanding of the recombinase structure, has therefore been adopted for Cre, Flp, and C31 Int. A typical procedure employs an in vivo test substrate, which is designed so that recombination at the sites under investigation alters the expression of an observable marker gene, such as a fluorescent protein or the lacZ‐encoded ‐fragment of ‐Gal (e.g., by excision of the gene, separating it from its promoter; Fig. 1.5). The substrate is established in the organism of choice, then challenged by introduction of DNA encoding a “library” of recombinase mutants (which may be generated by any appropriate method, such as error‐ prone PCR). Cells containing active mutants are then selected (e.g., by a change in color [Santoro and Schultz, 2002; Sclimenti et al., 2001]). A variation in the procedure, which facilitates the isolation of active mutants, is to combine the recombinase gene and the test construct in one plasmid (Buchholz and Stewart, 2001). Recombinase mutants obtained in the first cycle of mutagenesis‐ selection may then be used as the starting point for further cycles, either selecting for increased recombination efficiency on the same site or activity on a site that contains further changes from the wild‐type sequence. Mutants obtained by mutagenesis‐selection procedures, which can recombine at sites at which there are some differences compared with the wild‐ type site, generally have activity on the latter (normal) site (i.e., they have

Figure 1.5. An example of an assay for recombinase activity. Excision separates the reading frame of a marker gene (in this example green fluorescent protein, GFP) from its promoter, abolishing expression of the gene. Cells in which recombination has occurred can thus be detected (in this case, by loss of fluorescence). See text for further details.

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relaxed specificity). Therefore, some groups have developed methods to select, either simultaneously or sequentially, for activity on a variant site and loss of activity on the wild‐type site (Buchholz and Stewart, 2001; Voziyanov et al., 2002). A frequent elaboration of the mutagenesis‐selection procedure is to use “DNA shuffling,” a method that allows the generation of random combinations of a number of existing mutants (Stemmer, 1994), along with further mutagenesis; this potentially speeds up the selection process (Buchholz and Stewart, 2001; Santoro and Schultz, 2002; Sclimenti et al., 2001; Voziyanov et al., 2002, 2003). Impressive changes to the sequence specificities of Cre, Flp, and C31 Int have been achieved by these methods (see references previously cited), and specific activity at certain natural sequences in mammalian genomic DNA has been demonstrated (Buchholz and Stewart, 2001; Sclimenti et al., 2001; Thyagarajan et al., 2001). However, the strategy is not yet applicable to the targeting of sequences other than those that have been shown to be pseudosites or whose sequence is quite similar to the recombinase’s proper site. One might predict that, as more “specificity mutants” are identified, this approach would highlight the recombinase residues that are most important for sequence recognition. Further mutagenesis could then focus on these residues. Currently, it seems that many residues in Cre and Flp, along the whole length of the primary amino acid sequence, contribute to specificity. Some residues do appear as “hot spots” for mutations in the altered‐specificity variants, but as yet there is no clear pattern in the published data that would allow a more rational approach to the creation of new variants (Hartung and Kisters‐Woike, 1998; Voziyanov et al., 2003).

IX. STRUCTURE‐BASED STRATEGIES: Z‐RESOLVASES Crystallography of the serine recombinase resolvase reveals a modular structure. The 140‐residue N‐terminal domain, which contains all residues known to be involved in catalysis, is structurally and spatially distinct from the 40‐residue C‐terminal “helix‐turn‐helix” domain that is the primary determinant of sequence‐specific DNA binding (Fig. 1.3; Abdel‐Meguid et al., 1984; Grindley, 2002; Yang and Steitz, 1995). The two domains are connected by a short linker sequence that associates with the DNA minor groove. Resolvase and its very close relative Tn3 resolvase have been extensively studied in vitro (Grindley, 2002). The length of the tripartite recombination site (res; 114 bp), together with strong selectivities for supercoiling and pairs of sites that are in direct (“head‐to‐tail”) repeat on the same DNA molecule, renders these enzymes unsuitable for most of the envisaged applications of site‐specific recombinases. However, recent studies have led to “hyperactive” resolvase variants that


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recombine two copies of a short dimer‐binding site (the 28 bp site I of res) and no longer require supercoiling or directly repeated sites (Arnold et al., 1999; Burke et al., 2004). These variants might be put to the same kinds of uses as Cre and Flp. Mutant versions of relatives of Tn3/ resolvase have also been shown to act at simple dimer‐binding recombination sites without the need of additional factors (Johnson, 2002). Some minor alterations in sequence selectivity of this group of serine recombinases have been made by mutation, or by substituting the DNA‐binding domain with that of another member of the group (see Section VII). However, it became apparent that the sequence specificity of hyperactive resolvase variants might be more radically altered by replacing the DNA‐binding domain with one from an unrelated protein. The zinc‐finger DNA‐binding domain of the mouse transcription factor Zif268 was an especially attractive choice for this purpose. The Zif268 DNA‐binding domain is small (~90 amino acids), is monomeric in its functional form, and recognizes a short (9‐bp) sequence with high specificity. Its structure in a complex with DNA has been solved (Elrod‐Erickson et al., 1996; Pavletich and Pabo, 1991). Significantly, it is the focus of a campaign, involving a number of research groups, to create engineered zinc‐finger proteins, which can recognize any defined short DNA sequence (Beerli and Barbas, 2002; Pabo et al., 2001). Variants of the Zif268 domain that recognize some specific sequences in mammalian and viral genomes have already been created (Reynolds et al., 2003). The domain is composed of three similar zinc‐finger modules, each recognizing 3–4 bp of its 9‐bp target sequence. In each finger, only a few residues make base‐specific contacts with the DNA. It is therefore possible to simplify the task of selection for recognition of a new sequence by focusing on one finger at a time, and on the most important residues for recognition (Beerli and Barbas, 2002). The so‐called “phage display” technique has been adapted for the efficient selection of Zif268 domain variants that bind a chosen DNA sequence with very high affinity (Pabo et al., 2001), and other methods are being developed to select for high specificity (Hurt et al., 2003). These Zif268 domain variants with novel sequence specificity can act as artificial transcription factors, repressing or activating transcription from promoters close to their binding sites (Reynolds et al., 2003). They can also be used to tether an enzyme to a specific site on DNA. Novel chimeric nucleases have been created in which a nonspecific DNA cleavage domain is linked to a Zif268‐derived site‐specific DNA‐binding domain (Chandrasegaran and Smith, 1999). Cleavage mediated by the DNA‐bound chimeric enzyme occurs selectively at the chosen recognition site, and this can increase the in vivo efficiency of HR at that site (Bibikova et al., 2002, 2003; Porteus and Baltimore, 2003). Chimeric recombinases (“Z‐resolvases”) in which the catalytic domain of a hyperactive Tn3 resolvase variant is linked to the DNA‐binding domain of

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Figure 1.6. Cartoon of a Z‐site þ Z‐resolvase complex. Two 9 bp sequences (boxes with three arrows) are each recognized by a 3‐zinc‐finger DNA‐binding domain (dark grey shape) derived from that of Zif268. The 22 bp intervening sequence (hatched) interacts with the dimerized catalytic domains of a mutant version of Tn3 resolvase (light grey ovals) (see text for details). The Zif268 domain and the resolvase catalytic domain are linked by a short peptide (curved lines). The positions of strand breakage and rejoining are indicated by the staggered thin line.

Zif268 have been shown to recombine in vivo and in vitro specifically at “Z‐sites” (Akopian et al., 2003). Z‐sites consist of appropriately spaced pairs of 9‐bp sequence motifs recognized by the Zif268 domain, flanking a central sequence which is acted upon by the catalytic domains (Fig. 1.6). Each Z‐site binds a dimer of Z‐resolvase. The Z‐resolvase/Z‐site specificity was very high, and no activity on the natural target sequence of the catalytic domain (res site I) was detected. The rate of recombination was shown to depend on structural features of the Z‐resolvase and on attributes of the Z‐site sequence. The distance between the two 9‐bp motifs recognized by the Zif268 domains was critical, 22 bp being optimal. The reasons for this distance requirement are unknown as yet. The length and sequence of the peptide linking the two domains of Z‐resolvase also affected activity. In principle, any sequence of about 40 bp might be regarded as a potential Z‐site. Its ends could be recognized by variant Zif268 domains, and its central sequence could be cut and rejoined by resolvase catalytic domains. However, useable sequences might be relatively scarce. Thirteen basepairs of the original 28‐bp Tn3 res site I sequence were retained at the centre of all the Z‐sites tested so far (Fig. 1.6). Although current evidence suggests that resolvase contacts only a few basepairs of this sequence (Hatfull et al., 1988; Yang and Steitz, 1995), very different central sequences might be unsuitable. Sequences resembling that of the centre of res site I are likely to be the best targets, especially those with a central TATA motif, because these 4 bp contain the bonds that are broken and rejoined by resolvase. The 2‐bp “overlap” sequence in the TATA motif (AT) is palindromic, so a TATA‐centered Z‐site does not


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have a polarity. A substrate with two of these sites could recombine to give both excision and inversion products (see Section IV and Fig. 1.2B). Polarity can be restored by an asymmetric overlap sequence (e.g., AC), which prevents ligation of two left half‐sites or two right half‐sites (Stark et al., 1991). A pair of recombining sites must have the same overlap sequence; if not, recombination is blocked because the products would contain mismatched basepairs. Because resolvase assembles on DNA as a dimer, two Zif268 domain variants might have to be created, one to bind to each “half‐site” of a chosen genomic sequence (Fig. 1.6). In vivo targeting of a heterodimeric enzyme, with each subunit being fused to a different Zif268 domain variant, has a precedence in the studies on chimeric nucleases (see earlier section; Bibikova et al., 2003; Porteus and Baltimore, 2003). More generally, up to four Z‐resolvases, each with the same catalytic domain but a different Zif268 domain variant, could theoretically be used to recombine between two different natural Z‐sites (e.g., to excise a chosen segment of genomic DNA). However, strong recognition of just one half‐site by one subunit of a Z‐resolvase homodimer can suffice to target recombination (Akopian, A., unpublished results). Therefore, it is likely to be advantageous to use rare sequences as targets for each of the Zif268‐derived domains to minimize reactions at sequences other than the chosen Z‐site. The as‐yet hypothetical task of selecting a new site specificity for a Z‐resolvase can be split into parts, because of the enzyme’s modular nature. Zinc‐ finger domain variants that recognize the outer sequences of the Z‐site, and catalytic domains with optimal activity on the central sequence, would be created separately. Candidate recombinases targeted to the full chosen sequence would then be assembled by linking the selected domains. Enhancement of recombination activity on the chosen site could be achieved by mutagenesis‐ selection, as described in the Section VIII.

X. TARGETING TRANSPOSITION TO SPECIFIC SEQUENCES The problem of targeting transposition to specific genomic sites is somewhat different from the analogous problem for site‐specific recombination. A recombinase must be redirected from its natural site to a new target sequence, at which it would normally be inactive. In contrast, transposases act specifically at their cognate transposon ends but typically can insert them into many target sequences; the problem is to direct insertion to a chosen target site and ideally prevent insertion at any others. Whereas a “designer” site‐specific recombinase is generally intended to have activity at a single new site, transposase activity can potentially be targeted to larger regions of genomic DNA. One option is to “tether” the transposase to a specific binding site (e.g., by using an attached DNA‐binding domain), in order

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to favor transposition nearby. Alternatively, the transposase may be redesigned so as to interact with a specific DNA‐binding protein, so transposition occurs in regions of genomic DNA where the target protein is present. This strategy echoes the behavior of some natural retroviral integrases, which interact similarly with endogenous DNA‐binding proteins (Bushman, 2003; Sandmeyer, 2003). The published studies are all based on retroviral integrases, but similar approaches could be adopted for classical transposases. Fusions of HIV integrase with the DNA‐binding protein LexA (Goulaouic and Chow, 1996; Katz et al., 1996), Zif268 (Bushman and Miller, 1997), or E2C (Tan et al., 2004), and of Moloney murine leukemia virus integrase with Sp1 (Peng et al., 2002), have all been studied—the idea being that tethering the transposition intermediate to an appropriate binding site in the DNA will enhance insertion nearby. However, the results have been rather discouraging; the extent of integration near the target site was only a few times higher than that expected if target choice is random. Recent work by Voytas’ group (Zhu et al., 2003) showed how the yeast retrotransposon Ty5 is naturally targeted to heterochromatin by interaction with the protein Sir4p but will also insert close to artificially introduced ectopic Sir4p binding sites. Furthermore, the integrase protein can be redesigned so as to interact with other DNA‐binding protein partners. Target selection by some natural site‐specific DNA transposons also involves protein–protein interactions (e.g., Tn7; Craig, 2002b), but so far there have been no attempts to retarget them to new insertion sites. Despite these advances, it seems likely that competing transposon insertion at random sites will continue to be a problem until strategies that restrict transposase activity to the chosen locus are devised.

XI. GENERAL CONSIDERATIONS IN APPLICATIONS OF SITE‐SPECIFIC RECOMBINASES AND TRANSPOSASES A. Recombinase and transgene “delivery” In current applications of site‐specific recombination, DNA encoding the recombinases(s) and cognate recombination site(s) is either transfected or is preintegrated in the genome of the organism under study. Targeting of natural genomic sequences would normally still require transfection of DNA encoding the modified recombinase(s) along with any foreign sequences to be integrated. Recombinase‐based gene therapies would therefore have to surmount the same DNA delivery problems as all other gene therapies (Pfeifer and Verma, 2001). Of course, the difficulties are considerably less if transfection can be carried out


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in vitro or “ex vivo,” as would be the case for most nontherapeutic applications. Site‐specific recombination may be promoted by direct introduction of recombinase protein into cells (Baubonis and Sauer, 1993). It might even be practicable to introduce protein–DNA complexes in which the recombinase or transposase proteins required for targeted integration are already bound to the DNA (Goryshin et al., 2000). These ambitious approaches would avoid any undesirable side effects of prolonged production of recombinase from a transgenic expression construct (see Section D).

B. Chromatin Sequences that are inaccessible due to chromatin structure are likely to be poor targets for any recombinase‐based system. There is no obvious solution to this problem, although some enzymes may prove to be better than others in reaching protected sequences. Chromatin structure might also have effects on the sequence specificity of site‐specific recombinases or transposases (Portlock and Calos, 2003).

C. Reversibility Many site‐specific recombinases (e.g., Cre and Flp) promote recombination between two identical sites, so there are also two identical sites in the products. The reaction is therefore reversible (Kilby et al., 1993; Fig. 1.2B). The back reaction can be minimized if the recombinase is present only transiently (Baubonis and Sauer, 1993; Kilby et al., 1993). Some site sequences favor the forward reaction (Bouhassira et al., 1997; Hoess et al., 1986; Thomson et al., 2003), so it may be possible to choose pairs of sites for which this is the case. Certain recombinases, such as the phage integrases, recombine nonidentical sites, and their reactions can be essentially unidirectional (Groth and Calos, 2003). Similar problems might arise with DDE transposase‐mediated integration. The same transposase might promote excision of the integrated DNA. Such problems might be alleviated by strategies similar to those mentioned in Section A (e.g., transient availability of the transposase).

D. Nonspecific reactions All recombinases have the potential to cause “collateral damage” if they act at sequences other than the intended targets, and there is evidence that this happens in vivo. Cre can recombine at a number of pseudo‐loxP sites in the human and mouse genomes, which are quite divergent from the standard (“wild‐ type”) loxP sequence (Thyagarajan et al., 2000). Similar illegitimate recombination events, due to overexpression of Cre in transgenic mouse cells, can lead to

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growth inhibition, male sterility, cell‐cycle arrest, and DNA damage (Adams and van der Weyden, 2001; Loonstra et al., 2001; Schmidt et al., 2000). Such nonspecific activity would be very undesirable and potentially dangerous in gene therapy applications. The dangers of untargeted integration were tragically highlighted by two cases of leukemia in children participating in a trial for gene therapy of X‐linked severe combined immunodeficiency (X‐SCID). The therapeutic transgene was introduced ex vivo into cells that had been temporarily removed from the patients, by infection with a modified retrovirus whose integration was not targeted. Some of the retroviral integration events that ensued activated a nearby gene, resulting in oncogenesis (Kohn et al., 2003). Minimization of risk of analogous genetic damage would entail optimization of target site specificity, cell type‐specific recombinase gene delivery, cell type‐ specific promoters, and temporary recombinase expression. The tendency for retargeted recombinases to have relaxed specificity compared with their parent enzyme might restrict their applicability. Development of methods to select for restoration of high specificity is therefore most important. Of course, therapeutic modification of gene expression might be achieved in many circumstances by targeting but not altering DNA sequences (e.g., with DNA‐binding proteins or triplex‐forming oligonucleotides) (Uil et al., 2003).

XII. PROSPECTS AND CONCLUSIONS We still have a long way to go, but what will we be able to do when we have created efficient and highly specific “designer recombinases?” Perhaps the most exciting (but distant) prospects are in the field of gene therapy, where we can hope to bring about safe and efficient integration of transgenes at chosen genetic locations that support optimal expression. We might also knock out genes, or alter their expression levels, by integration of DNA sequences at suitable positions. Complete removal of a gene or other segment of DNA by recombinase‐ mediated excision will be very demanding technically because sites at both ends of the segment would have to be targeted by different versions of the recombinase. However, the task may be simpler in some special cases where an undesirable DNA segment is flanked by similar or identical sequences—the most obvious of these being the integrated proviral DNA of LTR retroviruses such as HIV. In the field of biotechnology, we can hope to improve and simplify procedures for making transgenic organisms for production of valuable proteins. Integration could be targeted to loci where gene expression will be high and only in the desired tissues (e.g., to a mammalian casein gene‐expression site, for production of proteins in milk).


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The uses of designer recombinases will not be restricted to mammals; similar strategies could be applied in any organism. In view of the concerns about introduction of transgenic DNA into food plants and wild species, the possibility that genetic changes at specific sites might be brought about by injecting designer recombinase proteins into cells, without any foreign DNA at all, may be appealing. Finally, the uses of these systems in experimental genetics, as tools for the specific genetic modification of laboratory organisms, are limited only by the scientists’ imagination.

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Pannell, D., and Ellis, J. (2001). Silencing of gene expression: Implications for design of retrovirus vectors. Rev. Med. Virol. 11, 205–217. Pavletich, N. P., and Pabo, C. O. (1991). Zinc finger‐DNA recognition: Crystal structure of a ˚ . Science 252, 809–817. Zif268‐DNA complex at 2.1 A Peng, W.‐J., Chang, C.‐M., and Lin, T.‐H. (2002). Target integration by a chimeric Sp1 zinc finger domain‐Moloney murine leukemia virus integrase in vivo. J. Biomed. Sci. 9, 171–184. Pfeifer, A., and Verma, I. M. (2001). Gene therapy: Promises and problems. Ann. Rev. Genomics Hum. Genet. 2, 177–211. Porteus, M. H., and Baltimore, D. (2003). Chimeric nucleases stimulate gene targeting in human cells. Science 300, 763. Portlock, J. L., and Calos, M. P. (2003). Site‐specific genomic strategies for gene therapy. Curr. Opin. Mol. Therapeut. 5, 376–382. Reynolds, L., Ullman, C., Moore, M., Isalan, M., West, M. J., Clapham, P., Klug, A., and Choo, Y. (2003). Repression of the HIV‐1 50 LTR promoter and inhibition of HIV‐1 replication by using engineered zinc‐finger transcription factors. Proc. Natl. Acad. Sci. USA 100, 1615–1620. Sandmeyer, S. (2003). Integration by design. Proc. Natl. Acad. Sci. USA 100, 5586–5588. Santoro, S. W., and Schultz, P. G. (2002). Directed evolution of the site specificity of Cre recombinase. Proc. Natl. Acad. Sci. USA 99, 4185–4190. Sauer, B. (2002). Chromosome manipulation by Cre‐lox recombination. In “Mobile DNA II” (N. L. Craig, R. Craigie, M. Gellert, and A. M. Lambowitz, eds.), pp. 38–58. ASM Press, Washington, DC. Schmidt, E. E., Taylor, D. S., Prigge, J. R., Barnett, S., and Capecchi, M. R. (2000). Illegitimate Cre‐ dependent chromosome rearrangements in transgenic mouse spermatids. Proc. Natl. Acad. Sci. USA 97, 13702–13707. Schneider, F., Schwikardi, M., Muskhelishvili, G., and Droge, P. (2000). A DNA‐binding domain swap converts the invertase Gin into a resolvase. J. Mol. Biol. 295, 767–775. Sclimenti, C. R., Thyagarajan, B., and Calos, M. P. (2001). Directed evolution of a recombinase for improved genomic integration at a native human sequence. Nucl. Acids Res. 29, 5044–5051. Shaikh, A. C., and Sadowski, P. D. (2000). Chimeras of the Flp and Cre recombinases: Tests of the mode of cleavage by Flp and Cre. J. Mol. Biol. 302, 27–48. Smith, K. (2001). Theoretical mechanisms in targeted and random integration of transgene DNA. Reprod. Nutr. Dev. 41, 465–485. Stark, W. M., Grindley, N. D. F. F., Hatfull, G. F., and Boocock, M. R. (1991). Resolvase‐catalysed reactions between res sites differing in the central dinucleotide of subsite I. EMBO J. 10, 3541–3548. Stemmer, W. P. C. (1994). DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution. Proc. Natl. Acad. Sci. USA 91, 10747–10751. Tan, W., Zhu, K., Segal, D. J., Barbas, C. F., III, and Chow, S. A. (2004). Fusion proteins consisting of human immunodeficiency virus type I integrase and the designed polydactyl zinc finger protein E2C: Direct integration of viral DNA into specific sites. J. Virol. 78, 1301–1313. Thomson, J. G., Rucker, E. B., III, and Piedrahita, J. A. (2003). Mutational analysis of loxP sites for efficient Cre‐mediated insertion into genomic DNA. Genesis 36, 162–167. Thyagarajan, B., Guimaraes, M. J., Groth, A. C., and Calos, M. P. (2000). Mammalian genomes contain active recombinase recognition sites. Gene 244, 47–54. Thyagarajan, B., Olivares, E. C., Hollis, R. P., Ginsburg, D. S., and Calos, M. P. (2001). Site‐specific genomic integration in mammalian cells mediated by phage phiC31 integrase. Mol. Cell Biol. 21, 3926–3934. Uil, T. G., Haisma, H. J., and Rots, M. G. (2003). Therapeutic modulation of endogenous gene function by agents with designed DNA‐sequence specificities. Nucl. Acids Res. 31, 6064–6078.


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Human Clinical Trials of Plasmid DNA Vaccines Margaret A. Liu* and Jeffrey B. Ulmer{ *Transgene S.A., 11, rue de Molsheim, 67082 Strasbourg Cedex, France { Chiron Corporation, Emeryville, California 94608

I. Background A. Historical perspective on DNA vaccines B. Mechanism of action II. Development of DNA Vaccines for Clinical Trials A. Manufacturing of plasmid DNA B. Qualification of plasmid DNA product C. Safety testing of DNA vaccines III. Human Clinical Trials A. Vaccines for infectious diseases B. Vaccines for cancer IV. Limitations of DNA Vaccine Potency V. Prospects and Conclusion References

ABSTRACT This article gives an overview of DNA vaccines with specific emphasis on the development of DNA vaccines for clinical trials and an overview of those trials. It describes the preclinical research that demonstrated the efficacy of DNA vaccines as well as an explication of the immunologic mechanisms of action. These include the induction of cognate immune responses, such as the generation of cytolytic T lymphocytes (CTL) as well as the effect of the plasmid DNA


0065-2660/05 $35.00 DOI: 10.1016/S0065-2660(05)55002-8

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upon the innate immune system. Specific issues related to the development of DNA as a product candidate are then discussed, including the manufacture of plasmid, the qualification of the plasmid DNA product, and the safety testing necessary for initiating clinical trials. Various human clinical trials for infectious diseases and cancer have been initiated or completed, and an overview of these trials is given. Finally, because the early clinical trials have shown less than optimal immunogenicity, methods to increase the potency of the vaccines are described. ß 2005, Elsevier Inc.

I. BACKGROUND A. Historical perspective on DNA vaccines It is now well established that plasmid DNA encoding a foreign protein can be expressed in situ, induce immune responses directed against the expressed protein, and elicit protective immune responses against the pathogen from which the protein was derived (Donnelly et al., 1997; Gurunathan et al., 2000). However, the initial reports on DNA vaccines were met with skepticism followed by considerable enthusiasm, as the technology proved to be quite facile and robust in a variety of laboratories and in vivo systems. Because viruses have evolved quite complex and specific mechanisms to deliver their genetic content to cells and because DNA is easily degraded in vivo, it was thought unlikely that DNA or RNA alone would be capable of transfecting cells in vivo. Moreover, because of the complexity of priming cellular immune responses (specifically processing and presentation of antigens leading to major histocompatibility complex [MHC] class I‐restricted CTL), simple administration of plasmid DNA was thought unlikely to be capable of generating the desired CTL responses. Nevertheless, DNA vaccines were shown to be capable of inducing both humoral and cellular immunity (i.e., both antibodies and CTL, along with T helper cells needed for the generation of both of the former responses) (Davis et al., 1993; Robinson et al., 1993; Tang et al., 1992; Ulmer et al., 1993; Wang et al., 1993; Xiang et al., 1994). The CTL responses were capable of providing immunity, which was protective against challenge of animals with a strain of virus that was different from the strain from which the gene was taken (Ulmer et al., 1993). These observations opened the door for a number of studies in different disease models in which DNA vaccines were able to induce immune responses and protective or therapeutic effects. Another development was the use of a so‐called ‘gene gun’ to propel microscopic gold beads coated with plasmid DNA into the skin, where the encoded protein was likewise produced with the subsequent generation of antibody and T cell responses against the protein (Fynan et al., 1993; Tang et al., 1992).

2. Human Clinical Trials of Plasmid DNA Vaccines

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The DNA vaccines used were simply bacterial plasmids, which utilized a promoter element active in mammalian cells rather than bacteria. Other common elements included a transcriptional terminator and a selectable marker that enabled the production of the plasmids in transformed bacteria. Thus, the concept of recombinant protein vaccines was taken one step earlier, by focusing on the production of the bacterial plasmid rather than on the encoded protein. One advantage of focusing on the plasmid as the product, is the generic nature of producing plasmids (i.e., the process for manufacturing the plasmid is relatively independent of the gene). The general approaches to produce plasmids for clinical usage are discussed in later section, but it is clear that having a generic manufacturing process irrespective of the vaccine product would greatly facilitate the development of many new vaccines. This is in contrast to the challenges of producing a live attenuated virus or expressing then purifying a recombinant protein; each of which would present formidable obstacles that must be otherwise uniquely overcome for each new product. Vaccines are considered to be the most successful medical intervention in the history of mankind. However, limitations with traditional vaccine approaches led scientists in search of new technologies for targets such as HIV, malaria, tuberculosis, and cancer. One key hurdle was to develop means of inducing robust cellular immunity, specifically CTL. These immune cells play an important role in the containment and clearance of tumors and cells infected with viruses or intracellular bacteria. Cytolytic T lymphocytes may be particularly important for protection against pathogens that induce chronic infections, such as HIV. The mechanism for the induction of CTL requires that the antigen be synthesized within cells of the vaccinated individual; delivery of an exogenous protein, such as the case with inactivated virus and recombinant protein vaccines, usually does not induce CTL responses. Live viruses effectively induce CTL, but the use of attenuated live viruses for certain pathogens, such as HIV, is considered too risky because of the possibility of reversion of the vaccine strain to the virulent and wild‐type strain. Thus, a technology, such as DNA vaccines, which could effectively induce protective CTLs and provide in vivo cross‐strain protection, in addition to inducing antibodies, provided a new approach for a variety of disease targets.

B. Mechanism of action The use of DNA vaccines has helped to elucidate two important aspects of the mechanism of action of immune priming (e.g., presentation of antigens by MHC class I molecules for priming CTL responses and the role of innate immunity in the generation of cognate immune responses against the antigen encoded by the DNA vaccine). First, as noted in earlier section, it is necessary for an antigen to be endogenously produced in a cell, preferably a professional antigen‐presenting

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cell (APC), in order to make the protein enter the MHC Class I processing pathway for presentation to, and priming of, naı¨ve CD8þ lymphocytes. If the antigen is delivered exogenously, or produced in a cell that lacks certain features (such as co‐stimulatory molecules found on professional APCs), CTL responses are not effectively generated. Since muscle cells are not APCs, the generation of CTL responses after intramuscular (IM) injection of DNA vaccines was surprising. The explanation may lay in observations by several groups that proteins encoded by DNA vaccines could be transferred from their site of synthesis (i.e., myocytes or keratinocytes) to professional APCs for presentation by MHC molecules (Cho et al., 2001; Corr et al., 1996, 1999; Fu et al., 1997; Kumaraguru et al., 2000; Ulmer et al., 1996). This phenomenon of cross priming may also account for induction of CTL against antigens on tumor cells and after infection of nonAPCs by viruses (Huang et al., 1994; Le Bon et al., 2003). Second, a key factor in the immunogenicity of DNA vaccines proved to be the bacterial DNA of the plasmid itself. Initially, the plasmid was thought to be simply the blueprint for the encoded protein and was considered immunologically inert. However, because DNA plasmids are derived from bacteria they stimulate the innate immune system by interacting with Toll‐like receptor 9 (TLR9) (Hemmi et al., 2000) and the resultant nonspecific immunity can augment the antigen‐specific immune response directed against the encoded protein (Klinman et al., 1997; Sato et al., 1996).

II. DEVELOPMENT OF DNA VACCINES FOR CLINICAL TRIALS The early successes of DNA vaccines in animal models provided the impetus to test this vaccine technology in human clinical trials. However, since this type of vaccine had never before been administered to humans, several formidable hurdles needed to be overcome to produce them, as well as to assure their safety and quality: (1) bacterial plasmids needed to be manufactured at scales never before attempted, (2) assays were required to measure the quality of this novel vaccine, and (3) certain safety evaluations not normally tested for conventional vaccines were needed (e.g., integration into host chromosomal DNA). This section will summarize the key development activities leading up to human testing of DNA vaccines.

A. Manufacturing of plasmid DNA Most plasmid DNA vaccines used to date contain the following basic elements: (1) an origin of replication for efficient propagation in Escherichia coli, (2) an antibiotic resistance gene for growth selection, (3) a strong promoter to drive expression in eukaryotic cells, (4) a polyadenylation termination sequence, and


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(5) a gene insert coding for the antigen of interest. In addition, immunostimulatory CpG motifs normally present in bacterial DNA contribute to vaccine potency via stimulating the innate immune system, as described in an earlier section. Two other types of plasmid vectors include those that do not contain antibiotic resistance genes, such as those utilizing repressor titration (Williams et al., 1998), and those encoding an alphavirus RNA replicon rather than a discrete gene (Berglund et al., 1998; Hariharan et al., 1998; Leitner et al., 2003). Both of these types of vectors have entered human clinical trials. For manufacturing purposes, it is desirable to have a system, which stably produces plasmids at high copy number. These characteristics facilitate the downstream processing steps and purification of the plasmid. The ColE1 origin of replication present in pUC‐based plasmids is commonly used, since yields of 30 mg of plasmid per liter of culture can be achieved. Other factors contributing to high plasmid yield include the host E. coli strain, growth medium and conditions of fermentation. For example, E. coli strains that produce relatively low levels of nucleases can result in greater plasmid stability during processing. The manufacture of plasmid DNA has a potentially distinct advantage over production of other types of vaccines. Unlike other processes, such as those for recombinant proteins where specific procedures and conditions are required for each product, all plasmid DNA vaccines are physically similar to one another. Hence, methods for their production and purification are essentially generic. The majority of plasmids produced during fermentation of E. coli have a supercoiled topology (i.e., a coiled coil). Other minor forms are produced as a consequence of nicking one of the chains resulting in relaxed circles, nicking of both chains to produce linear plasmids, and denaturation. It is generally believed that supercoiled plasmids are the most active of these forms, based on transcription in vivo (Kano et al., 1981; Sekiguchi and Kmiec, 1989). However, both relaxed circle and linear plasmids are immunogenic as DNA vaccines in animals (unpublished observations). Stability studies in vitro have shown that supercoiled plasmids are sequentially converted to relaxed circles, linear plasmids, fragments, and oligomers (Evans et al., 2000). Therefore, it is desirable to have supercoiled plasmids to increase the shelf life of the product. After fermentation, plasmid DNA at best accounts for only 1% of the total cell mass. Thus, procedures are required for the release of plasmids from the cells and their separation from other cellular constituents. In the laboratory, this is effectively accomplished using alkaline lysis of the cells, precipitation of nonplasmid material by SDS, and sedimentation of the precipitate to yield an aqueous preparation of plasmid. A high degree of purity can be achieved by a subsequent CsCl gradient centrifugation step. Some of these steps are neither practical nor acceptable for use at large scale for preparation of human clinical materials. For example, the CsCl gradient centrifugation step was eliminated

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due to the impracticalities of centrifugation and the toxicity of the materials used. Instead, anionic exchange chromatography is often utilized to take advantage of the negative charge of the plasmid DNA. Prazeres et al. (2001) reviewed these steps involved in plasmid DNA purification. The demand for plasmid DNA has resulted in a fledgling industry for the contract manufacture of both research‐grade material for animal studies and cGMP‐grade product for human clinical trials.

B. Qualification of plasmid DNA product Purified plasmid DNA products should conform to the following specifications (Robertson and Griffiths, 2001; Smith and Klinman, 2001). The product should appear clear and colorless by visual inspection. The identity of the plasmid can be confirmed by nucleotide sequence, PCR for plasmid‐specific sequences and/or restriction enzyme digest patterns by gel electrophoresis. As mentioned in an earlier section, the form of the plasmid should be predominantly circular (preferably supercoiled), as judged by gel electrophoresis. Potency of the product can be quantified by expression of the transgene in transient transfection of cells in vitro and/or by induction of immune responses against the transgene in an appropriate animal model. Plasmid DNA concentration is measured by absorbance at 260 nm. Purity of the product is assessed by the presence of residual proteins, chromosomal DNA, RNA, and endotoxin. The levels of these impurities should fall below specified limits. Finally, sterility is measured by standard microbiological assays.

C. Safety testing of DNA vaccines Standard preclinical evaluation of vaccine safety includes systemic and local reactogenicity, histopathology, and toxicity in appropriate animal models. Due to the unique nature of plasmid DNA vaccines, the following additional safety tests are performed. First, although hypothetical at this point, integration of DNA plasmids into host chromosomal DNA is possible. Therefore, quantitative assays to assess such integration events have been developed. These are based on physical separation of high molecular weight chromosomal DNA from plasmids by gel electrophoresis, followed by PCR for plasmid‐specific sequences. This approach can detect a single integration event in 150,000 cellular nuclei (Martin et al., 1999; Nichols et al., 1995). So far, little or no integration has been observed in animal models (Kang et al., 2003; Ledwith et al., 2000b; Manam et al., 2000). Second, because certain types of anti‐DNA antibodies can be pathogenic and cause autoimmune disorders (e.g., systemic lupus erythematosus), the potential for induction of such antibodies by plasmid DNA vaccines must be evaluated. This possibility, though, is also hypothetical, as


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humans are frequently exposed to bacterial DNA without apparent consequence. So far, there have been no reports of induction of pathogenic anti‐ DNA antibodies by DNA vaccines (Parker et al., 1999, 2001). Third, the biodistribution and persistence of plasmid DNA vaccines are evaluated in animal models. Unlike vaccines based on proteins or inactivated organisms, DNA vaccines result in the in situ production of antigen. Therefore, it is important to know the specific cell types that may produce the antigen and how long this production may persist. This is accomplished by PCR analyses of various tissues over time for the presence of plasmid‐specific sequences. After IM injection, typical plasmid DNA vaccines have been shown to be present in several tissues for a period of days but are quickly cleared (Hanke et al., 2002; Parker et al., 1999). Thereafter, plasmids persist in the injected muscle for many weeks, but slowly disappear over time. These results are consistent with a lack of integration and loss of plasmid by nuclease digestion and cellular turnover. Based on this type of safety profile, many DNA vaccines have entered human clinical trials.

III. HUMAN CLINICAL TRIALS Plasmid DNA has been evaluated in a variety of human clinical trials for potential use as prophylactic vaccines, as immunotherapy for cancer, as immunomodulators for asthma/allergy, and as gene therapy for chronic diseases. As with any new vaccine, the initial phase I trials are designed to test the safety of the candidates, particularly because this technology had been used never before for direct human administration. An overview of the types of clinical trials will be given here to highlight the issues for usage and efficacy of plasmid DNA in humans.

A. Vaccines for infectious diseases The ability of DNA vaccines to induce cytolytic T cell responses and the potential for the technology to be amenable for manufacture and distribution on a global scale and in developing countries, led to the rapid clinical testing of DNA vaccines for infectious diseases such as HIV, malaria, and hepatitis B. The earliest clinical trials were with HIV DNA vaccines conducted in individuals already infected with HIV, followed by extension to volunteers uninfected with HIV. These studies, as well as later studies for other indications, demonstrated that DNA vaccines are well tolerated and safe. There has been no evidence of integration of DNA into host chromosomes, autoimmunity, or immunologic tolerance (Comerota et al., 2002; Conry et al., 2002; Epstein et al., 2002; Klencke et al., 2002; MacGregor et al., 2000; Swain et al., 2000; Tacket et al., 1999; Weber et al., 2001).

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While the overall clinical immune responses have been less potent than expected based upon the preclinical results in animal models, there have been notable exceptions, and more trials utilizing higher DNA doses, alternative protocols, and novel formulations have resulted in greater immunogenicity. Different trials have resulted in different results in terms of the types of immune responses generated, perhaps as a reflection of the different DNA constructs, immunization protocols, and patient populations. For example, several studies demonstrated the generation of CTL responses following DNA immunization (Calarota et al., 1998, 2001; Roy et al., 2000). In another study, although some HIV seronegative patients immunized with DNA encoding env/rev were able to generate CD4 T helper responses including the production of IFN‐gamma in an ELISPOT assay (MacGregor et al., 2002), no CD8 responses were observed. Interestingly, in HIV infected patients, a DNA vaccine was able to induce antibody and CTL responses against certain viral antigens against which no detectable antibodies or CTL had existed prior to vaccination despite a high preexisting viral load (Calarota et al., 1998, 1999). A slightly different approach utilizing a DNA vaccine encoding consensus HIV clade A Gag p24/p17 proteins fused to a linear sequence of CTL epitopes tested alone and in conjunction with a viral vector (modified vaccinia Ankara, MVA) in a DNA prime‐vector boost approach was shown capable of generating responses directed against many of the CTL epitopes, as measured by IFN‐gamma ELISPOT (Mwau et al., 2004). These HIV trials have demonstrated that immune responses (primarily T cells) can be induced or boosted in humans by DNA vaccines. Although most of these trials were not designed to address the effectiveness of vaccination against HIV disease, it is very likely that significant enhancements will be required to elicit protective immune responses by DNA vaccines. DNA vaccines encoding malarial antigens have been shown capable of generating CD8þ CTL, even against epitopes restricted by more than one HLA haplotype in the same person (Wang et al., 1998). A follow up study confirmed the generation of CTL by a malaria DNA vaccine (Le et al., 2000; Wang et al., 2001). However, no antigen‐specific antibody responses were detected in either trial. The strategy of employing a heterologous gene delivery system in combination with DNA vaccines is in clinical evaluation and has been found promising. For example, although DNA and MVA vectors by themselves are both capable of generating IFN‐gamma antigen‐specific immune responses, the responses are substantially greater if the DNA is given first, followed by MVA (McConkey et al., 2003; Moorthy et al., 2003). Another clinical trial utilizing the prime‐boost approach has demonstrated the capability of a recombinant protein to act as a booster vaccine for DNA (Epstein et al., 2004; Wang et al., 2004). Clinical trials of malaria vaccines offer the possibility of measuring protective efficacy by challenge with live parasites. Preliminary results with the DNA prime‐MVA boost approach have shown protection, as seen by delayed parasitemia following sporozoite challenge with a different strain of malaria (McConkey et al., 2003).


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Studies utilizing a hepatitis B DNA vaccine highlight an alternative delivery technology, employing a device to propel gold beads coated with DNA into the epidermis of vaccinated individuals. Using this approach, a DNA vaccine was shown capable of inducing both antibodies and cellular responses in hepatitis‐ naı¨ve individuals (Roy et al., 2000). Although the magnitude of the antibodies induced by this DNA vaccine was not as high as that elicited by the licensed recombinant protein vaccine, a potential advantage of the DNA vaccine is the ability to induce T cell responses. Both CD4þ and CD8þ cells were elicited by the DNA vaccine, with the helper cells being of the Th1 phenotype (i.e., secreting IFN‐gamma). In a more challenging clinical situation, patients who had either not responded to a recombinant protein vaccine or whose response had not persisted, were immunized with plasmid DNA‐coated gold beads. The DNA was capable of inducing an antibody response in 12 of these individuals despite their previous failure to respond adequately to the licensed vaccine (Rottinghaus et al., 2003). Taken together these data hold promise for potential use of a hepatitis B DNA vaccine in nonresponder individuals and for immunotherapy to clear infection.

B. Vaccines for cancer Plasmid DNA has likewise been clinically evaluated as a therapeutic vaccine approach for cancer. These DNA vaccines have encoded tumor antigens, such as viral epitopes from transforming viruses (Klencke et al., 2002), self‐antigens that are expressed on tumors (Mincheff et al., 2000; Rosenberg et al., 2003; Tagawa et al., 2003), and tumor‐specific antigens (Timmerman et al., 2002). While most trials of prophylactic DNA vaccines are injected i.m. or intraepidermally (via particle bombardment), the cancer immunotherapeutic vaccines have also been delivered intranodally (Tagawa et al., 2003) and intradermally (Mincheff et al., 2000) as well. One study utilized a formulation of the DNA vaccine in an effort to facilitate the delivery of the DNA to APCs after IM injection (Klencke et al., 2002). Most of these cancer DNA vaccines have shown limited effectiveness as an immunotherapeutic intervention, possibly due to a relatively immunocompromised status of the cancer patients. These clinical trials of DNA vaccines for infectious diseases and cancer have proven the principle that immune responses can be generated in humans, but highlight the need for increased potency if this vaccine technology is to be fully effective.

IV. LIMITATIONS OF DNA VACCINE POTENCY Effective vaccines typically consist of three key components. First, the antigen is required to elicit a specific memory immune response, so that upon subsequent exposure to the pathogen a rapid and specific immune response is mounted to

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prevent and/or clear infection. For DNA vaccines, the antigen is expressed by the plasmid in cells in situ. Based on reporter gene expression, only very small amounts of protein are produced (Wolff et al., 1990). Thus, it is important that the DNA vaccine vectors used are optimized for protein expression. Elements of the plasmid that contribute to the level of protein expression include the promoter, enhancer, and polyadenylation terminator. In addition, the nucleotide sequence and codon usage of the gene insert can play significant roles in transcription and translation (Haas et al., 1996; Qiu et al., 1999; zur Megede et al., 2000). Second, an immune stimulus or adjuvant is usually needed to maximize the immunogenicity of the vaccine. Insight into the nature of innate immune signaling has revealed that adjuvants generally interact with receptors (e.g., TLRs) present on immune cells. This interaction results in rapid activation of cells leading to cytokine production and an increase in antigen presentation capacity. Thus, when this occurs in the presence of an antigen, the result is an enhancement of antigen‐specific immune responses. DNA vaccines appear to have a built‐in adjuvant in the form of immunostimulatory CpG motifs, which are known to utilize TLR9 (Hemmi et al., 2000). The presence of these motifs is thought to play a role in the potency of DNA vaccines (Klinman et al., 1997; Sato et al., 1996), suggesting that the correct number and placement of CpG within the plasmid are important. Third, a vaccine delivery system can protect the vaccine from degradation, facilitate cellular uptake, target specific cells or tissues, and ensure that the antigen and adjuvant are delivered together. Conventional vaccines based on live attenuated organisms have all of these three elements contained within the vaccine. However, highly purified subunit vaccines, such as recombinant proteins and naked DNA vaccines, generally lack adjuvants and delivery systems and require that they be added back to the vaccine. Given that naked DNA vaccines have no inherent means of efficiently transfecting cells, delivery systems may offer the greatest opportunity for improvement. Intramuscular injection of naked plasmid DNA results in the transfection of very few cells in situ (Dupuis et al., 2000; Wolff et al., 1990). Based on PCR analysis, it was estimated that only a few thousand copies of plasmid are functionally retained within cells of the injected muscle (Ledwith et al., 2000a). Therefore, approximately only one in ten million plasmids are effectively delivered into the nuclei of cells. Limitations on the cellular delivery of plasmid DNA appear to be manifested in several areas, including distribution of the vaccine solution within the injected tissue, cellular uptake, and intracellular delivery to the nucleus (Dupuis et al., 2000). Transfection of APCs was found to be particularly inefficient after i.m. injection. These observations suggest two basic strategies to enhance the delivery, hence potency, of DNA vaccines. First, increased distribution of the DNA vaccine within the injected tissue should allow a greater number of cells to be available for transfection. Technologies to


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physically deliver DNA plasmids to tissues, such as the gene gun, needle‐free devices, electroporation, and hydrostatic pressure, result in broader expression and enhanced vaccine potency (Wells, 2004). Second, expression of DNA vaccine antigens by APCs is known to be an effective means to induce immune responses (Manickan et al., 1997; Timares et al., 1998). Hence, targeting plasmid DNA specifically to APCs should increase vaccine potency. The effectiveness of particle‐based formulations of plasmid DNA (Little et al., 2004; Singh et al., 2000), which facilitate DNA uptake by and antigen expression within phagocytic APCs (Denis‐Mize et al., 2000, 2003), is consistent with this hypothesis. These delivery systems and formulations have in many cases shown marked increase in DNA vaccine potency in nonhuman primates (O’Hagan et al., 2001; Otten et al., 2004) and some of these technologies have entered human clinical trials (Klencke et al., 2002).

V. PROSPECTS AND CONCLUSION DNA vaccines have been widely utilized in immunological studies and as a reagent for a variety of laboratory studies. Early clinical studies have demonstrated that the most likely strategies just now entering the clinic, such as novel delivery systems or combinations with other gene‐delivery vectors will possibly, be the means to boost the potency of the DNA vaccines. Despite the added complications of such second generation approaches, the inherent capabilities and characteristics of DNA vaccines (i.e., the ability to induce CTL and Th1 type helper responses, the potential advantages of their manufacture, and the platform nature of the technology) continue to make this a technology with significant appeal.

References Berglund, P., Smerdou, C., Fleeton, M. N., Tubulekas, I., and Liljestrom, P. (1998). Enhancing immune responses using suicidal DNA vaccines. Nat. Biotechnol. 16, 562–565. Calarota, S., Bratt, G., Nordlund, S., Hinkula, J., Leandersson, A. C., Sandstrom, E., and Wahren, B. (1998). Cellular cytotoxic response induced by DNA vaccination in HIV‐1‐infected patients. Lancet 351(9112), 1320–1325. Calarota, S. A., Kjerrstrom, A., Islam, K. B., and Wahren, B. (2001). Gene combination raises broad human immunodeficiency virus‐specific cytotoxicity. Hum. Gene. Ther. 12(13), 1623–1637. Calarota, S. A., Leandersson, A. C., Bratt, G., Hinkula, J., Klinman, D. M., Weinhold, K. J., Sandstrom, E., and Wahren, B. (1999). Immune responses in asymptomatic HIV‐1‐infected patients after HIV‐DNA immunization followed by highly active antiretroviral treatment. J. Immunol. 163(4), 2330–2338. Cho, J. H., Youn, J. W., and Sung, Y. C. (2001). Cross‐priming as a predominant mechanism for inducing CD8(þ) T cell responses in gene gun DNA immunization. J. Immunol. 167(10), 5549–5557.

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3 Genetic and Environmental

Influences on Antisocial Behaviors: Evidence from Behavioral–Genetic Research Terrie E. Moffitt Institute of Psychiatry, King’s College London, SE5 8AF United Kingdom and University of Wisconsin, Madison, Wisconsin 53706

I. Appraising the Quantitative Behavioral–Genetic Evidence Base A. The number of studies has increased B. Techniques for statistical analysis are more sophisticated C. More studies use large sample sizes D. More is known about how twins and adoptees represent the population E. Behavioral–genetic studies have measured antisocial behaviors with different kinds of valid methods F. Data are now available from many different types of behavior–genetic designs G. Looking for a sturdy finding II. Estimating the Relative Influences of Genes and Environments A. Genes influence approximately 50% of the population variation in antisocial behaviors B. Environmental factors shared by family members influence about 20% of population variation in antisocial behaviors C. Environmental factors experienced uniquely by individuals influence about 20–30% of population variation in antisocial behaviors D. Summary of quantitative genetic findings


0065-2660/05 $35.00 DOI: 10.1016/S0065-2660(05)55003-X

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III. Do the Omnibus Estimates of Genetic and Environmental Influences Always Apply, or Do These Influences Vary Under Different Conditions? A. Is there a sex difference in the genetic influence on antisocial behaviors? B. Are there historical cohort differences in the genetic influence on antisocial behaviors? C. Is there a genetic influence on physical violence? D. Is there a genetic influence on the psychopath? E. Are genetic influences involved when antisocial behavior co‐occurs with other forms of psychopathology, such as hyperactivity? F. Can antisocial experience influence genes? IV. Testing Developmental Theory of Antisocial Behavior A. Stronger genetic liability may be associated with life‐course‐ persistent than adolescence‐limited antisocial behaviors V. Testing Hypotheses About Environmental Causation VI. Testing the Hypothesis of Interaction Between Genes and Environments A. Adoption studies of latent G E B. Adoption studies of latent G measured E C. A twin study of latent G measured E D. Studies of measured G measured E; testing a measured gene E. Research implications of the nil main effect of the MAOA polymorphism on behavior F. Strategy for future G E studies using measured genes VII. The Way Forward Acknowledgments References

ABSTRACT This article reviews behavioral–genetic research into human antisocial behavior. The focus is on studies of antisocial behavior that have been leading the way in investigating environmental and genetic influences on human behavior. The first generation of studies, which provided quantitative estimates attesting that genes and environments each influence about half of the population’s variation in antisocial behaviors is interpreted. Then how behavioral–genetic methods are being applied to test developmental theory and to detect environmental causes

3. Genetic and Environmental Influences on Antisocial Behaviors

43

of antisocial behavior is illustrated. Evidence for interactions between genes and the environment in the etiology of antisocial behavior is also examined. The article ends by envisioning future work on gene–environment interplay in the etiology of antisocial behavior. ß 2005, Elsevier Inc. Despite many years of assiduous efforts to eliminate it, antisocial behavior is still a problem. Approximately 20% of people in the developed world experience victimization by perpetrators of violent and nonviolent illegal behavior each year (U.S. Bureau of Justice Statistics, 2002). The World Report on Violence and Health (WHO, 2002) tallies the staggering burden of mortality, disease, disability, and compromised well‐being brought about by perpetrators of family violence and other violent crimes. Behavioral science needs to achieve a complete understanding of the causes of antisocial behavior to provide an evidence base for effectively controlling and preventing antisocial behavior. Research into genetic and environmental influences is making great strides toward uncovering the root causes of antisocial behavior. Studies are revealing which risk factors are causes, not just correlates. Studies are testing for effects of measured candidate genes. Studies are sorting out how our genotypes sway our susceptibility to environmental causes and how our environments rule the behavioral expression of our genotypes. Studies are refining the antisocial phenotype, uncovering a serious and persistently antisocial subgroup that appears to be more genetically influenced than ordinary antisocial behavior. Journalists have drawn public attention to certain families that seem to contain far more than their share of criminal family members across several generations (Butterfield, 1996, 2002). This familial concentration of crime has been confirmed as a characteristic of the general population (Farrington et al., 1996, 2001; Rowe and Farrington, 1997). In general, fewer than 10% of the families in any community account for more than 50% of that community’s criminal offenses. The family concentration of antisocial behavior could be explained by a genetic influence on antisocial behavior, but it could just as easily be explained by nongenetic social transmission of antisocial behavior within families. Behavioral–genetics research disentangles genetic from nongenetic aspects of familial transmission. Behavioral genetics also has methods to put genetic and nongenetic influences back together again in a systematic and controlled way, to work out how they jointly influence behavior. Behavioral genetics has been rapidly moving beyond the initial question of whether behavior is heritable (Dick and Rose, 2002; Kendler, 2001) to apply its methods to a broad array of causal questions about developmental processes influencing behavior. Given that virtually all behavior and certainly antisocial behavior must be the product of interplay between genes and environments, progress toward understanding cause–effect processes depends on studies not only to separate

44

Terrie E. Moffitt

genetic from nongenetic influences but also studies that can reunite them to observe their interplay. Here, interplay refers to processes in which genes and environments conferring risk for psychopathology co‐occur (gene–environment correlation) and jointly influence the probability that psychopathology will develop (gene environment interaction). Section I of this article critiques the methodological quality of the behavioral–genetic research that has apportioned genetic versus environmental influences on antisocial behavior. How good is the evidence base? Section II summarizes the quantitative estimates of genetic and environmental influences on antisocial behavior resulting from this evidence base, and explains what the findings mean. Section III queries genetic findings with respect to sex differences, cohort effects, violence, the psychopath, antisocial behavior that co‐ occurs with mental disorders, and assortative mating. Section IV illustrates ways that behavioral–genetic designs are being applied to test developmental theory about antisocial behavior. Section V explains how behavioral–genetic designs are newly being used to distinguish risk factors for antisocial behavior that are bona fide environmental causes. Section VI examines interactions between genetic and environmental causes of antisocial behaviors. Section VII puts forward directions for future research.

I. APPRAISING THE QUANTITATIVE BEHAVIORAL–GENETIC EVIDENCE BASE Tables 3.1 to 3.3 bring together all quantitative behavioral–genetic studies of antisocial behaviors, inclusively defined, that were available as at 2004. Reviews up to the mid‐1990s concluded that evidence was accumulating that genetic factors influence which individuals in the population exhibit antisocial behaviors (Carey, 1994; Carey and Goldman, 1997; Gottesman et al., 1997; McGuffin and Thapar, 1998; Miles and Carey, 1997; Raine, 1993; Walters, 1992). The literature of published behavioral–genetic studies of antisocial behaviors has expanded dramatically since those reviews appeared, and there have been six notable improvements in the quality of research into the genetic and environmental architecture of antisocial behaviors.

A. The number of studies has increased To date, more than 100 quantitative genetic studies of antisocial behaviors have been published from more than 60 different genetically informative samples, totaling more than 77,000 families. The last columns of Tables 3.1 to 3.3 show that more than one‐third of the studies have been published after 1994.

Table 3.1. Early Childhood: Estimates of Genetic and Environmental Influences on Population Variation in Antisocial Behavior, from Different Types of Behavioral–Genetic Studies of Young Children Environment estimates Heritability estimate

Common

Unique þ error

Measure of antisocial behavior

Data source

Number of families

Age of probands

Nation/ sample

Authors

Year of publication

Twins reared together design 34%

32%

34%

60%

20%

20%

49–75%

0–22%

25–29%

58%

0%

42%

82%

0%

18%

69%

0%

31%

76%

0%

24%

42%

0%

58%

61%

0%

39%

CBCL Externalizing CBCL Externalizing CBCL Externalizing Physical aggression CBCL Externalizing/ opposition observations/ Berkeley Puppet Conduct Problems CBCL Externalizing CBCL Externalizing Berkeley Puppet Conduct Problems Oppositional behavior at home

Mother

260

2–3

USA, CO

Parents

1358

3

The Netherlands

Mother

3620

2–3

Mother

4562

19 months

Composite mother/teacher/ observer/self

1116

5

The Netherlands Canada, Quebec UK, E‐risk

Mother

1116

5

UK, E‐risk

Teacher

1116

5

UK, E‐risk

Self

1116

5

UK, E‐risk

Observer

1116

5

UK, E‐risk

Schmitz et al. van den Oord et al. van der Valk et al. Dionne et al.

1995 1996

1998 2003

Arseneault et al.

2003

Arseneault et al. Arseneault et al. Arseneault et al.

2003

Arseneault et al.

2003 2003

2003

(Continues)

Table 3.1. (Continued) Environment estimates Heritability estimate 23%

Common 10%

Unique þ error 67%

Measure of antisocial behavior Disruptive behaviors on videotape, blind observers

Data source Observation in a game of SNAP!

Number of families 1116

Age of probands 5

Nation/ sample UK, E‐risk

Authors

Year of publication

Hughes et al.

2002

Adoptees design No studies Twins reared apart design No studies

Note 1: Studies having fewer than 75 families are excluded from the table. We make an exception to this rule for studies having rare design features (twins reared apart or observational measurement). Note 2: When variance estimates are presented as ranges (e.g., 45–65%), this is usually because the original publication provided estimates separately for males and females. In a few cases, ranges are estimates provided separately for successive waves of a longitudinal study, or for different subscales of a measure. Note 3: Certain samples are represented on more than one row of the table, but each row presents data from a different cohort, a different age or a different measurement source or instrument.

Table 3.2. Middle Childhood to Adolescence: Estimates of Genetic and Environmental Influences on Population Variation in Antisocial Behavior, from Different Types of Behavioral–Genetic Studies of School‐Aged Children and Adolescents Environment estimates Heritability estimate

Common

Unique þ error


Data source

Number of families

Age

172 total

Adolescence

USA 2, Germany 1, Japan 3, UK 1

5–11 7

USA, CO USA, CO

Nation/sample

Authors

Year of publication

Twins reared together design Very low in 7 studies

Not estimated

Not estimated

Delinquency

Official

0% 60%

45% 12%

35% 28%

Hit Bobo doll Bullying

Observer Adult

0–42%

19–69%

31–39%

Rutter antisocial scale

Adult

205

13

UK, London children

57% 51%

22% 28%

21% 21%

CBCL Externalizing CBCL Externalizing

Adult Adult

399 181

4–18 7–15

0%

61%

39%

Adult

198

8–16

37–46%

46–50%

6–12%

Rutter antisocial scale CBCL Externalizing

USA, CO USA, Western Reserve UK, Cardiff

Adult

780

5–9

Norway

13–38% 25–66%

46–62% 4–42%

16–25% 29–33%

Adult Adult

1264 1197

8–16 8–16

USA, VA USA, Virgnia

25–74% 57–65%

0–44% 22–50%

25–77% 6–12%

CBCL Externalizing Rutter antisocial scale Conduct disorder CBCL Externalizing

Adult Adult

1412 1048

8–16 12–15

USA, VA Norway

60%

30%

10%

CBCL Externalizing

Adult

720

9–18

USA, NEAD

85 87

DiLalla and Gottesman, 1989, Table 1; Raine, 1993, Table 5 Plomin et al. O’Connor et al. Stevenson and Graham Schmitz et al. Edelbrock et al. Thapar and McGuffin Gjone and Stevenson Silberg et al. Silberg et al. Eaves et al. Gjone and Stevenson Deater‐ Deckard et al.

1934– 1977

1981 1980 1988

1995 1995 1996 1997 1994 1996 1997 1997b 1997

(Continues)

Table 3.2. (Continued) Environment estimates Heritability estimate

Common

Unique þ error

62–77% 50–76% 70–77% 29–69%

4–12% 0–18% 0% 0–27%

18–27% 24–32% 23–30% 31–44%

50%

18%

70%


Data source

Number of families

Age

Nation/sample

Authors

Year of publication

Adult Adult Adult Adult

1022 501 492 125

7–9 8–16 8–12 7–11

Sweden UK USA, Missouri UK

Eley et al. Eley et al. Hudziak et al. Kuntsi et al.

1999 1999 2000 2000

32%

CBCL Aggression CBCL Aggression CBCL Aggression Conners’ conduct probs CBCL Externalizing

Mother

1116

7

UK, E‐risk

2003

1%

28%

CBCL Externalizing

Teacher

1116

7

UK, E‐risk

7%

36%

57%

Self

1116

7

UK, E‐risk

63%

29%

8%

Dominic‐R DSM conduct problems Olweus scale

Unpublished data from the author Unpublished data from the author Arseneault et al.

1412

8–16

USA, VA

Simonoff et al.

1995

61%

14%

25%

BPI antisocial

405

10–18

USA, NEAD

14%

34%

1506

11

USA, MN

90%

0%

43%

524

17

USA, MN

Krueger et al.

2001

62%

0%

0%

Conduct disorder symptoms Broad externalizing disorder spectrum Conduct problems scale

O’Connor et al. Burt et al.

1998c

52%

Self/adult composite Self/adult composite Self/adult composite Self/adult composite Self/adult composite

5–17

Scourfield et al.

2004

80%

No report

No report

2004

6%

52%

Self/adult composite Self

Hicks et al.

42%

Broad externalizing disorder spectrum MMPI psychopathy

CaStANET, Wales and North England USA, MN USA,Boston

Gottesman

1966

1109

542 152

17 (plus parent) 14–18

2003

2005

2001

14–74%

0–41%

26–45%

70% 25–36% 81%

No report 0–42% No report

No report 52–77% No report

47–54% 18% 35%

0–13% 26%

40–46% 56%

61%

0%

39%

7%

31%

62%

23%

No report

No report

71%

0%

29%

34%

30%

37%

38%

0%

61%

31–36%

10–17%

52–54%

Sibhip design 55%

14%

31%

32%

5%

63%

Socialization aggression Delinquency Conduct disorder Conduct disorder items

Self

326

Self Self Self

99 1412 81

Socialization Delinquency Conduct disorder items Conduct disorder items

Self Self Self

Adults recall conduct symptoms Adults recall conduct symptoms Adults recall conduct symptoms Adults recall conduct symptoms Adults recall conduct symptoms Antisocial to parent

18 13–18 8–16 Adolescence

USA, merit scholars USA, Ohio USA, VA UK, Cardiff

Loehlin and Nichols Rowe Eaves et al. McGuffin and Thapar Taylor et al. Taylor et al. Young et al.

1976

381 486 334

16–18 16–18 12–18

USA, MN USA, MN USA, CO

2000 2000a 2000

Self

740

13–21

Miles et al.

2002

Self

3226

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