Locus control subregions conferring integration-site independent transgene expression abstract of the disclosure

The invention encompasses a locus control subregion that possesses chromatin opening domain activity, the activity conferring reproducible activation of tissue-specific expression on a linked transgene to a non-physiological level when the transgene is integrated in single copy in the genome of a host cell.

FIELD OF THE INVENTION 
The invention relates to the expression of heterologous genes in eukaryotic 
host cells and transgenic animals. 
BACKGROUND OF THE INVENTION 
Locus Control Regions (LCRs) (Grosveld et al., Cell 51:975-985, 1987), also 
known as Dominant Activator Sequences, Locus Activating Regions or 
Dominant Control Regions, are responsible for conferring tissue specific, 
integration-site independent, copy number dependent expression on 
transgenes integrated into chromatin in host cells. The discovery and 
characterization of LCRs are described in co-pending U.S. Ser. No. 
07/920,536, filed Jul. 28, 1992, assigned to the same assignee, the 
complete disclosure of which is hereby incorporated by reference. First 
discovered in the human globin gene system, which was prone to strong 
position effects when integrated into the chromatin of transgenic mice or 
mouse erythroleukaemia (MEL) cells (Magram et al., Nature 315:338-340, 
1985; Townes et al., EMBO J. 4:1715-1723, 1985; Kollias et al., Cell 
46:89-94, 1986; Antoniou et al., EMBO J. 7:377-384, 1988), LCRs have the 
ability to overcome such position effects when linked directly to 
transgenes (Grosveld et al., supra). Numerous LCRs have been defined in 
the art, including but not limited to the .beta.-globin and CD2 LCRs 
(European Patent Application 0 332 667), the macrophage-specific lysozyme 
LCR (Bonifer et al., 1985), and a class II MHC LCR (Carson et al., Nucleic 
Acids Res. 21, 9:2065-2072, 1993). 
The complete .beta.-globin LCR comprises four DNase I hypersensitive sites 
(HS) on a 20 kbp fragment that is too large to be incorporated into 
retrovirus or adeno-associated virus (AAV) vectors designed for 
integration into the mammalian genome. Individual hypersensitive sites, in 
particular the 5'HS2 associated element, have been studied for the ability 
to regulate transduced globin genes (Novak et al., Proc. Natl. Acad. Sci. 
USA 87:3386-3390, 1990; Chang et al., Proc. Natl. Acad. Sci. USA 
89:3107-3110, 1992; Miller et al., Blood 82:1900-1906, 1993). However, it 
has proven to be difficult to obtain stable high-titer viruses bearing 
these sequences. 
When referring to the DNase I hypersensitive sites of the .beta.-globin 
LCR, care must be taken over the nomenclature used. Originally, the 
hypersensitive sites were numbered consecutively 1 to 4 working upstream 
from the globin genes by Tuan et al., (Proc. Natl Acad. Sci. USA 
86:2554-2558, 1985) and downstream towards the gene by Grosveld et al. 
(supra). In 1990, agreement was reached to use the nomenclature in which 
5'HS1 is closest to the globin genes and 5'HS4 is most distant from the 
genes. The GenBank numbering for HS2, HS3 and HS4 is GenBank 958-1714, 
GenBank 4248-5197, and GenBank 8486-8860, respectively. A number of 
publications dating back from around or before 1990 use the inverse 
nomenclature. 
Previous work demonstrated that each hypersensitive site of the human 
.beta.-globin locus control region confers a different developmental 
pattern of expression on the globin genes (Fraser et al., 1993, Genes & 
Development 7:106-113). HS3 was shown to be most active during the 
embryonic period, whereas HS4 showed the highest activity during the adult 
stage. Each of HS1 and HS2 drive equivalent levels, albeit low and high 
levels, respectively, of .gamma. or .beta. transgene expression throughout 
development. 
Previous work demonstrated that the 5' HS2 core region, i.e., a 215 bp 
fragment containing four putative transcription factor binding sites, 
functions in a concatemer of at least two copies but not when present as a 
single copy in transgenic mice to confer position independent expression 
of a linked transgene (Ellis et al., Eur. Mol. Biol. J. 12:127-134, 1993). 
Thus, two or more 5'HS2 cores may interact and cooperate with each other 
to open chromatin and enhance transcription, however, a single 5'HS2 core 
fails to activate expression from a linked .beta.-globin gene in 
single-copy founder transgenic mice. This failure of the single copy 
HS2/transgene construct to activate transgene transcription was 
demonstrated unequivocally using fully transgenic F.sub.1 offspring. No 
such data exists in the prior art for the HS3 or HS4 .beta.-globin LCR 
subregions. 
HS3 and HS4 have only been tested in founder (F.sub.0) animal tissue, that 
is, using embryo tissue that has been grown from injected eggs, which 
tissue can carry different copy numbers of a transgene in different cells. 
(Philipsen et al., 1993, EMBO J. 12:1077-1085; Pruzina et al., 1991, 
Nucleic Acids Res. 19;1413-1419). 
Studies using founder animal tissue are highly inconclusive with respect to 
transgene copy number because copy number cannot be determined 
definitively. Because a transgene integrates into the injected egg after 
the single cell stage, different tissues almost always contain different 
copy numbers (or no copies) of a transgene. Therefore, reliable data as to 
expression of an HS3/or HS4/transgene construct in single copy cannot be 
obtained using founder animals. There is no indication in founder animals 
of the extent to which the transgene has integrated into the animal's 
somatic tissues. For instance, a nominal copy number of two could indicate 
the presence of two copies of the transgene in each cell, or four copies 
in half of the cells, or eight copies in one quarter of the cells, and so 
on. In addition, because of the minute amount of embryo tissue available, 
e.g., fetal liver tissue, copy number analysis in founder animals is 
performed on tissues other than that used for analysis of the expression 
level of the transgene. However, true copy number can be determined 
reliably in F.sub.1 generation animals in which the transgene has been 
passed through the germ line by breeding of the founder animal. F.sub.1 
transgenic animals contain an equal number of copies of the transgene in 
each cell. 
When experiments are conducted on single-copy transgenic animals, it is 
found that many of the LCR fragments previously believed to confer LCR 
activity are incapable of satisfying the functional requirement of an LCR, 
namely the conferring of integration-site independent expression on a 
transgene, when present in a single copy. Clearly, such DNA elements are 
inappropriate for protocols where the use of single copy gene is 
desirable, essential or inevitable, as in the case of many virus-based 
delivery systems. 
Previously, it has been found that the full activity of an LCR appears to 
be obtainable only with complete LCR sequences. Thus, in the .beta.-globin 
LCR, only a construct containing the DNA sequences surrounding all four of 
the DNase I hypersensitive sites 1 to 4 confers tissue-specific, 
integration site independent, copy number dependent expression of a 
transgene at levels reflecting the level of expression of an equivalent 
endogenous gene. 
It is an object of the invention to provide for reproducible 
integration-site independent expression of a transgene in a mammal, 
particularly a human, when the transgene is integrated in single copy in 
the mammalian genome. 
Another object of the invention is to identify a sub-fragment of an LCR 
that reproducibly confers the chromatin opening activity of the LCR when 
present in single copy. 
Yet another object of the invention is to provide an LCR subregion which, 
when operatively associated with a transgene and integrated in single copy 
in a host cell genome, reproducibly confers integration-site independent, 
tissue-specific expression on the transgene. 
Another object of the invention is to provide for reproducible, 
tissue-specific, integration-site independent expression of a single copy 
transgene using gene transfer techniques that are limited with respect to 
the amount of DNA that is transferred to a host genome. 
SUMMARY OF THE INVENTION 
The invention is based on the discovery that the chromatin opening function 
of a Locus Control Region (LCR) is separable from other functions of an 
LCR, and may be carried on a portable subfragment of the LCR. The present 
invention is directed to gene therapy through delivery of a recombinant 
DNA vector containing a chromatin opening domain and a functional 
(expressible) gene to the cells of a patient, and expression of the gene 
in a position independent manner. 
The present invention accordingly provides a recombinant DNA vector 
comprising an LCR subregion comprising a chromatin opening domain operably 
linked to an expressible gene, and a carrier for introducing the vector 
into a host cell via transmembrane delivery. 
Preferably, the transmembrane delivery carrier will include one of a 
protein carrier as for cell-cell fusion, an antibody carrier, a liposomal 
carrier and viral carriers. More preferably, the carrier for vector 
delivery includes carriers which deliver the vector via the process known 
as receptor-mediated endocytosis, i.e., the use of a ligand capable of 
binding to a membrane receptor, the complex of which is taken into the 
cell via membrane invagination. 
A "chromatin opening domain" is characterized in that, when it is operably 
linked to a gene and integrated in single copy into the genome of a host 
cell, it reproducibly actuates tissue-specific expression of the gene to a 
non-physiological level, i.e., a level that is below the level of 
expression of a transgene that is operably linked to an equivalent fully 
functional LCR, independent of the site of integration of the gene in the 
genome. 
As used herein, "chromatin opening domain" refers to a region of DNA that 
is defined in terms of both its structure and function. Structurally, the 
region is defined in that it comprises a region of an LCR that encompasses 
at least one DNase I hypersensitive site when the region is in its native 
(i.e., naturally occurring) context in the chromosome; functionally, it is 
defined in that it possesses the property of actuating transcription of a 
linked gene independent of the integration site of the gene in a host cell 
genome. "Actuating transcription" refers to the ability of the domain to 
allow for activation of transcription of the linked gene. This process of 
actuating transcription is believed to involve the ability of the 
chromatin opening domain to render the region of chromatin encompassing 
the linked gene (or at least its promoter) accessible to transcription 
factors. An "expressible" gene refers to a gene and genetic control 
elements necessary for expression of the gene in a host cell. 
As will be understood by a person skilled in the art, a locus control 
region (LCR) is a region of DNA that confers copy number dependent and 
integration site independent expression of a linked gene (i.e., a gene 
with which it is associated). 
It has been observed that a fully-functional LCR directs expression of a 
transgene to a physiological level, i.e., a level equivalent to that 
observed for an equivalent endogenous gene. In contrast, an LCR subregion 
comprising a chromatin opening domain that is the subject of this 
invention does not achieve the full activity of an LCR but activates 
transcription at a non-physiological, i.e., lower level. Preferably, the 
lower level is less than or equal to about 80% of a physiological level; 
more preferably, less than about 60%; and most preferably, less than about 
45%. By "physiological level of a gene product" is meant a level of gene 
function at which a cell population or patient exhibits the normal 
physiological effects made by the presence of normal amounts of the 
encoded protein. Insufficient amounts of a gene product will result in 
deleterious or unwanted clinical symptoms, e.g., as are associated with a 
disease. 
Preferably, a domain of the invention is associated with, i.e., 
encompasses, a DNase I hypersensitive site in the genome of a cell. It is 
believed that the majority of LCRs will be found to be associated with 
DNase I hypersensitive sites in natural cell chromatin and moreover it 
appears that discrete elements of the LCR are marked by single 
hypersensitive sites within a cluster of sites which is associated with 
the presence of the complete LCR. 
A fully functional or complete LCR will, of course, include a chromatin 
opening domain. The ability to convert chromatin to an open conformation 
is a key feature of an LCR, particularly for those uses of the invention 
involving expression of single-copy transgenes. This function has been 
discovered to be separable from other functions of the LCR which act to 
confer physiological level expression. The chromatin opening DNA element 
of the invention is a region of DNA that transforms a closed chromatin 
structure into an open chromatin structure. 
A chromatin opening domain of the invention is in its native context part 
of a fully functional LCR. 
A chromatin opening domain of the invention also preferably restores the 
DNase I hypersensitivity found in the subregion in its native chromatin 
context to the subregion when integrated in a host cell genome. 
LCRs display tissue specificity in their behavior, and the chromatin 
opening domains of the invention possess equally tissue-specific 
characteristics. Therefore, the appropriate domain will be selected 
according to its tissue-specific characteristics, such that it will 
function to actuate transgene expression only in a desired tissue. For 
example, the .beta.-globin or macrophage-specific lysozyme LCRs are 
specific for a particular hematopoietic cell lineage. It will be apparent 
to those skilled in the art that appropriate LCR subregions may be 
selected for individual applications, as required. 
One of skill in the art would be able to determine if a chromatin opening 
domain exists in a region of DNA by cloning a region of DNA that is 
suspected of containing such a domain, linking the cloned region to a 
reporter gene to generate a construct, introducing the construct into a 
host cell, preferably of a mammal, and measuring RNA encoded by the linked 
gene (or a portion thereof) relative to the amount of RNA encoded by an 
endogenous gene that is under the control of an equivalent endogenous 
locus control region in the host cell. The cloned region of DNA will be 
determined to contain a chromatin opening domain if actuation of 
transcription of the linked gene is reproducibly obtained among host cell 
genomes containing single copy chromatin opening domain/transgene 
constructs. Typically, 3-5 single copy host cell genomes (e.g., transgenic 
animals) will be tested for transgene transcription in order to ascertain 
reproducible actuation of transcription. Moreover, the linked gene will 
express an amount of RNA that is less than about 80% (i.e., in the range 
of 1-80%), preferably less than about 60% or 45% of the amount of RNA that 
is expressed by a single copy of the endogenous gene. 
The invention thus also encompasses testing a region of a tissue specific 
locus for chromatin opening domain activity, such activity being defined 
herein as conferring tissue specificity of the locus from which the domain 
is derived and conferring position independent gene expression, the 
activity being conferred by a region of DNA that is smaller than the 
region identified as a locus control region (i.e., including an associated 
enhancer), and the level of transgene expression conferred being less than 
that conferred by the corresponding fully functional LCR. The testing 
construct includes a reporter gene, such as the .beta.-galactosidase 
reporter gene driven by the mouse heat shock promoter 68 (hsp 68). Other 
reporter genes are also contemplated according to this aspect of the 
invention, including but not limited to the luciferase gene. 
In one embodiment of this aspect of the invention, the .beta.-galactosidase 
reporter gene and the mouse heat shock promoter 68 are operationally 
associated with the .beta.-globin HS3 COD. Thus, the construct is 
introduced into cells of a lineage for which the tested COD is tissue 
specific, e.g., blood cells for the .beta.-globin HS3 COD, and expression 
of the reporter transgene is detected and optionally quantified in that 
tissue. 
The domains of the invention are typically much smaller than the 
fully-functional LCR. For example, the fully-functional .beta.-globin LCR 
is 20 kb in length, while the smallest domain identified which has the 
ability to confer an open chromatin conformation is 1.9 kb in length. This 
reduction in size is advantageous in that it allows for packaging of 
LCR-active constructs into available viral delivery systems. Many viral 
delivery systems for the delivery of genes encoding therapeutic products 
involve integration of transferred DNA in single copy in host cells. 
Moreover, use of single copy integrants reduces the risk of possible 
adverse effects on the host cell genome by ensuring that the number of DNA 
recombination events is kept to the absolute minimum. Packaging into 
alternative, non-viral delivery systems and vectors is also facilitated. 
The invention thus enables constructs having LCR activity which are 
considerably smaller than was previously possible using the entire LCR. A 
construct according to the invention is active when integrated in single 
copy number, thus providing reproducible long-term (stable) expression of 
a transgene. 
The invention further provides domains according to the invention for use 
in medicine. In particular, the domains of the invention are indicated for 
use in the manufacture of medicaments. For example, products such as human 
growth hormone or human factors VIII or IX may be made in a transgenic 
animal using a construct of the invention. 
Chromatin opening domains according to the invention may be found in 
association with a variety of LCR sequences. The LCR sequences are 
preferably of mammalian origin, but other vertebrate sequences, such as 
avian LCR sequences, are known. 
One detailed embodiment of the invention provides a chromatin opening 
domain that is associated with one of the four DNase I hypersensitive 
sites of the human .beta.-globin LCR in native chromatin. It has been 
discovered that the DNA sequence surrounding 5'HS3 in the human 
.beta.-globin LCR is responsible for reproducibly directing actuation of 
transcription of a linked transgene when the construct is present in 
single copy in transgenic mice, independent of the site of integration of 
the construct in the mouse genome. 
Preferably, the 5'HS3 construct comprises the 1.9 kb DNA sequence between 
the Hind III sites 14.3 to 16.2 kb upstream of the .epsilon.-globin gene. 
After having identified a DNA sequence responsible for conferring chromatin 
activation, as taught herein, it will be apparent to one skilled in the 
art that one can combine such a sequence with a chosen enhancer element to 
increase the expression level of a transgene. For example, use of a strong 
heterologous enhancer with a domain of the invention may lead to 
physiological-level expression of the transgene in the desired tissue 
type, independently of the site of integration of transgene in host cell 
chromatin. Strong enhancers are well-known in the art and include, e.g., 
the .beta.-globin HS2 or HS4 enhancers, the .alpha.-globin enhancer, and 
certain viral enhancers known in the art. Use of a regulatable enhancer, 
e.g., hormone-inducible enhancers such as steroid, especially 
glucocorticoid-induced enhancers, or viral enhancers, can provide further 
control over the expression of the transgene. Other suitable enhancer 
constructs are well known in the art and may be selected for their known 
properties. 
A construct according to the invention may include a chromatin opening 
domain in combination with a heterologous enhancer. The heterologous 
COD/enhancer combination allows one of skill in the art to choose a 
combination which will confer a desired level of transgene expression, 
i.e., that is not achievable using a homologous COD/enhancer combination. 
The heterologous COD/enhancer combination will achieve a level of 
transgene expression that is either less than or greater than the level of 
transgene expression achieved using a homologous COD/enhancer combination. 
As used herein, "greater than" or "less than" means at least 10% or 
preferably 20-25% greater than or less than the level of expression of a 
homologous COD/enhancer combination. 
Other heterologous enhancers useful according to this aspect of the 
invention include but are not limited to the human Cytomegalovirus (CMV) 
enhancer, the .alpha.-globin 40 kb enhancer, SV40 enhancers, adenovirus 
enhancers, immunoglobulin enhancers, and T cell receptor enhancers. In 
addition, the enhancer corresponding to the promoter and/or transgene 
present in the construct are useful according to the invention. 
Preferably, the .beta.-globin HS3 COD is combined with a heterologous 
enhancer to achieve a level of transgene expression that is different from 
the level of transgene expression achieved using the complete 
.beta.-globin LCR (i.e., consisting of HS1-HS4). 
In the case of the .beta.-globin 5'HS3-associated chromatin opening domain, 
in particular, a homologous COD/enhancer combination is particularly 
useful. That is, the 5'HS3 chromatin opening domain is combinable with the 
5'HS2-associated fragment, which is active as an enhancer in erythroid 
tissue. Although the 5'HS3 domain alone confers chromatin opening activity 
on an associated transgene, and thus confers a non-physiological level of 
transcription, as defined above, it has been discovered that a construct 
consisting essentially of 5'HS3 and 5'HS2 operationally associated with a 
transgene provides for tissue-specific, integration site-independent 
expression of the transgene that is higher than the level of transgene 
expression in erythroid tissue using the 5'HS3 region alone. 
The invention also encompasses vectors containing the LCR subregion 
chromatin opening domain, as described herein, and kits for reproducibly 
actuating expression of a transgene in a host cell, the kit containing DNA 
comprising an LCR subregion as described herein and container means 
therefore. 
The invention also provides for methods for conferring integration site 
independence on a transgene integrated in single copy in the genome of a 
host cell comprising operably linking a domain, as defined above, to the 
transgene. 
The transgene or expressible gene may be any desired gene, and is 
preferably a gene whose presence in a host cell corrects a genetic 
disorder. For example, globin-encoding genes, clotting factor-encoding 
genes, protein hormone-encoding genes, and ligand receptor-encoding genes. 
Particular reference is made to genes encoding proteins which have 
therapeutic utility, such as therapeutically useful proteins or ribozymes, 
or genes encoding anti-sense RNA. 
Therapeutically useful proteins include anti-viral agents and decoy 
proteins useful in the prophylaxis or treatment of viral disease, 
especially diseases such as AIDS, as well as proteins which act to 
supplement or replace natural proteins that are defective. The use of 
transgenes expressing therapeutically useful intracellular antibodies is 
envisaged (see, for example, WO93/12232; WO94/02610). 
The invention also provides a method for identifying a chromatin opening 
domain which comprises (a) providing a host cell containing a DNA 
construct in single copy, the construct comprising a candidate LCR 
subregion comprising a chromatin opening domain operably linked to an 
expressible reporter gene; (b) determining that the DNA construct 
reproducibly actuates transcription in the host cell by ascertaining 
transgene transcription for independent integration events in at least 3 
separate genomes; and also may include the step of (c) comparing the 
amount of RNA encoded by the reporter gene with the amount of RNA encoded 
by an endogenous gene that is operably linked to an equivalent complete 
LCR endogenous to the host cell, wherein the presence of an LCR subregion 
comprising a chromatin opening domain is indicated if the amount of 
reporter gene-encoded RNA is less than about 80% of the amount of RNA 
encoded by the endogenous gene. 
The invention also encompasses treatment of certain genetic diseases 
utilizing constructs according to the invention. For example, X-linked 
.gamma.-globulinemia is treated by introducing a construct according to 
the invention into pre-B cells and introducing the transfected pre-B cells 
into a patient afflicted with X-linked .gamma.-globulinemia. The construct 
includes the Bruton's kinase promoter and transgene operationally 
associated with a chromatin opening domain of the class II major 
histocompatibility complex (MHC) gene LCR. This chromatin opening domain 
will confer tissue-specificity corresponding to that of the full-length 
class II MHC LCR, and thus will direct transgene expression primarily in B 
or pre-B cells, and will also confer position-independent transgene 
expression of the full length LCR, but will not allow for expression of 
the transgene to the level conferred by the full length LCR. The reduced 
level of transgene expression conferred by the class II MHC chromatin 
opening domain will be less than about 60% of the level of expression of 
the transgene when associated with the full length corresponding LCR, will 
likely be less than about 40%, and may be on the order of about 10-25%. 
As used herein, a "pre-B cell" refers to an immune system cell as defined 
in The Leukocyte Antigen Fact Book, 1993, Barclay et al., Eds., Academic 
Press, Harcourt Brace, London. A pre-B cell is thus defined by Barclay et 
al. as possessing the following cellular markers: CD9, CD10, CD19, CD20, 
CD22, CD24, CD38, CD40, CD72, CD74, and is surface Ig negative. 
The invention also encompasses treatment of Gaucher's disease by 
introducing into a macrophage host cell a construct including the 
.beta.-glucocerebrosidase transgene whose expression is initiated by the 
lysozyme gene promoter. The construct will also include the chromatin 
opening domain of the macrophage-specific lysozyme gene LCR. The 
macrophage-specific lysozyme chromatin opening domain will retain the 
tissue-specificity of the full-length lysozyme LCR, and thus will direct 
transgene expression primarily in macrophages, and will also retain 
position-independent transgene expression of the full length LCR, but will 
not allow for expression of the transgene to the level conferred by the 
full length LCR. 
Preferably, the reduced level of transgene expression conferred by the 
macrophage-specific lysozyme chromatin opening domain will be less than 
about 60% of the level of expression of the transgene when associated with 
the full length corresponding LCR, will likely be less than about 40%, and 
may be on the order of about 10-25%. 
This construct is used to treat Gaucher's disease by introducing 
transfected macrophages into a patient afflicted with Gaucher's disease. 
Expression of the wild type transgene in a patient afflicted with 
Gaucher's disease should result in correction of the diseased state. 
As used herein, a "macrophage" refers to an antigen presenting, phagocytic 
cell as defined in The Leukocyte Antigen Fact Book, 1993, Barclay et al., 
Eds., Academic Press, Harcourt Brace, London. That is, a macrophage is 
defined in Barclay et al. as including the following cell surface markers: 
CD14, CD16, CD26, CD31, CDw32, CD36, CD45RO, CD45RB, CD63, CD71, and CD74. 
As used herein "macrophage" refers to either a resting cell or an 
activated cell, and thus may also possess the cell surface markers: CD23, 
CD25 and CD69. 
The invention also encompasses treatment of genetic or transmitted diseases 
utilizing a chromatin opening domain as described herein. For example, the 
CD2 chromatin opening domain may be used in conjunction with an 
expressible gene to treat a T-cell associated disorder or disease. A 
T-cell associated disease or disorder is treated by introducing into a 
T-cell a construct including the CD2 chromatin opening domain and a gene 
encoding a protein for expression in T-cells, e.g., and interleukin, such 
as IL-2, a growth factor, or a viral determinant, such as an HIV 
determinant. 
The invention further provides the use of a domain of the invention for the 
generation of transgenic mammals. In particular, the invention provides 
the use of such sequences for the generation of non-human transgenic 
mammals, which may be germ-line transgenic or somatic transgenics, 
especially transgenic mice, particularly for the purpose of drug 
development. 
The invention is defined with respect to the following terms and 
definitions. As used herein, a "fully functional" or "complete" LCR is 
able to direct expression of a linked gene (which is termed a transgene 
when integrated into a host cell genome) to a "physiological" level, i.e., 
a level that is about equivalent to that observed for the endogenous gene 
that is associated with an equivalent endogenous LCR in the host cell. 
"About equivalent" refers to at least 80%, and preferably 95-100% of the 
expression level of the endogenous gene. Therefore, a "physiological" 
level of transcription or gene expression refers to any level that is 
equal to or above about 80% of the level of expression of the endogenous 
host cell gene. 
A "fully functional" or "complete" LCR also refers to a region of DNA that 
includes all of the DNase I hypersensitive sites that are necessary to 
obtain at least 80-90%, and preferably 95-100%, expression of a linked 
gene when integrated into the genome of a host cell relative to a 100% 
expression level of a single copy of a gene that is under the control of 
an equivalent endogenous LCR in the host cell. 
An "equivalent endogenous LCR" is defined as a region of DNA that confers 
copy number dependence and integration site independence on substantially 
the same coding region that the test LCR is associated within its native 
context. This coding region or gene naturally occurs in the host cell in 
association with the equivalent endogenous LCR. An equivalent endogenous 
LCR may refer to the complete LCR from which the LCR subregion is 
obtained; e.g., the human .beta.-globin LCR could be considered an 
equivalent endogenous LCR for a human .beta.-globin LCR subregion. Thus, a 
human .beta.-globin LCR subregion may be tested in human erythroid cells 
with respect to the human .beta.-globin LCR in the same type of human 
cells. 
Alternatively, and often in practice, an equivalent endogenous LCR is the 
equivalent LCR that occurs in another species; e.g., where the host cell 
is a mouse cell and the introduced (test) LCR subregion and linked gene 
are the human .beta.-globin LCR and gene, respectively, an "equivalent 
endogenous LCR" will be the mouse .beta.-globin LCR and the endogenous 
gene will be the mouse .beta.-globin gene. Thus, a .beta.-globin LCR 
subregion may alternatively be measured by comparing the expression level 
of the human .beta.-globin gene relative to the expression level of the 
mouse .beta.-globin gene in mouse erythroid cells. The latter comparison 
is used only in those cases in which the two species of genes are 
expressed at equal levels in the two species of cells. The former 
comparison is made where equivalent gene expression levels are 
unobtainable across species. 
Although the test LCR is measured with respect to its equivalent endogenous 
LCR in the host cell, the linked gene may be but need not be the same 
coding region as the endogenous gene. For example, where the host cell is 
a mouse cell and the test LCR and linked gene are the human .beta.-globin 
LCR and the human CD2 gene, the test LCR/linked gene construct is measured 
in at least 3 transgenenic animals by comparing the amount of RNA coding 
for the linked gene (the CD2 gene in this example) to the amount of RNA 
coding for the mouse .beta.-globin gene. Thus, in this example, expression 
of the human CD2 gene is stated in a percentage that is relative to 
expression of the mouse .beta.-globin gene because both genes are under 
control of "equivalent", i.e., .beta.-globin LCRs. 
An "LCR subregion consisting essentially of a chromatin opening domain" is 
definable herein in its simplest terms as a region of DNA (that includes a 
chromatin opening domain) that is not a fully functional LCR in both 
structure and function. 
Structurally defined, it is a subregion of DNA that is shorter in length 
than the fully functional LCR. "Shorter in length" may mean, for example, 
equivalent to as much as 80-90% of the length of an LCR, i.e., a region of 
DNA that has been determined to confer a physiological level of transgene 
expression, as defined herein, or a length which is only 50% or as little 
as 10%, 25% or 40% of the length of the complete LCR. However, because the 
actual length of 20 an LCR chromatin opening domain subregion relative to 
the length of a "complete" LCR is not meaningful unless the minimum region 
of the "complete" LCR has been determined, a functional definition of this 
functional LCR subregion is also necessary for determining the presence of 
a chromatin opening domain. 
Functionally defined, an LCR subregion consisting essentially of a 
chromatin opening domain retains the natural ability of the complete LCR 
when integrated with a transgene as a single copy construct to confer 
tissue-specific gene expression on the associated transgene, but does not 
possess the complete LCR's ability to confer "physiological" level 
expression on the transgene, but rather confers a level of transcription 
on the transgene that is somewhat lower than the full transcriptional 
activity naturally conferred by a complete LCR. As used herein, a 
"non-physiological level" of expression or transcription of the transgene 
refers to a level of expression that is less than about 80%, may be less 
than 70%, and may even be a value in the range of 5-50%, or for example, a 
value in the range of 1%-45%, relative to the level of expression of the 
endogenous host cell gene (i.e., a gene that is associated with an 
equivalent endogenous host cell LCR, as defined above). Of course, the 
level of expression of the endogenous host cell gene or the gene 
controlled by the equivalent fully functional LCR is arbitrarily set at 
100%. 
An LCR subregion of the invention also retains the ability of the complete 
LCR to confer integration site independent expression on the transgene; 
however, such site-independence is reproducibly retained only in the sense 
that the subregion allows for at least some (e.g., at least about 5%) 
expression of the transgene regardless of the integration site of the 
transgene in the host cell genome. The actual level of transgene 
expression may vary from one site to the next (e.g., it may vary from 
1-45% among the different sites). Thus, as used herein, "reproducible" 
does not refer to an ability to confer a given level of transcription on a 
transgene, but rather to the ability to actuate transcription of a 
transgene independent of its integration site in a host cell genome. Thus, 
actuation of transgene expression is reproducible if the transgene is 
expressed in a minimum of three independent host cell genome integration 
events. An independent integration event may be represented, for example, 
by integration of a single construct into the genome of an F.sub.1 
generation transgenic animal, and thus three such events by three F.sub.1 
generation transgenic animals. 
At its minimum functional definition, a chromatin opening domain that is 
not a fully functional LCR is a recombinant DNA that is a subregion of an 
LCR that consists essentially of a chromatin opening domain in that it 
reproducibly actuates integration site-independent transcription of a 
linked transgene, when the domain and linked transgene are present in 
single copy in a genome, and is hypersensitive to DNase I or endogenous 
nuclease, but does not contain other functional elements that may be 
contained within an LCR, i.e., enhancer activity. Thus, another functional 
test for open chromatin domain activity according to the invention is 
DNase I hypersensitivity. As described hereinbelow, DNase I hypersensitive 
site mapping may be performed on nuclear DNA from a given tissue of a 
transgenic animal containing the candidate chromatin opening domain in 
single copy. The presence of a chromatin opening domain will be indicated 
by the presence of a DNase I hypersensitive site at the site of 
integration of the COD/transgene construct in the chromosome. 
"Enhancer" activity may be separated from "chromatin opening" activity even 
though both activities affect the expression level of the associated 
transgene. That is, enhancer activity boosts expression of the transgene 
but requires the presence of the chromatin opening domain. In contrast, 
chromatin opening domain activity allows for some transgene expression 
(i.e., at least 1%) that does not rise to physiological levels of 
expression in the absence of the enhancer element. The ability to convert 
chromatin to an open conformation is the key feature of an LCR subregion 
comprising a chromatin opening domain and refers to the ability of the 
subregion to reproducibly actuate a "non-physiological level" of 
expression of the linked transgene when the subregion/transgene construct 
is present in single copy in the host cell genome. 
Although the level of gene expression that is conferred by an LCR subregion 
containing a chromatin opening domain may for some such LCR subregions 
appear to be an exceedingly low level (e.g., 1-5%) relative to the level 
of expression of the endogenous gene that is associated with an equivalent 
endogenous LCR, it is according to another comparison a significant level 
of expression of the transgene. For example, if transgene expression is 
compared in a first construct containing the transgene and an LCR 
chromatin opening domain subregion and a second construct containing a 
different LCR subregion that does not contain a chromatin opening domain, 
for example an LCR subregion that contains only an enhancer, the LCR 
subregion that contains the chromatin opening domain will confer 
significantly higher (e.g., 10-fold greater) transgene expression than an 
LCR subregion lacking a chromatin opening domain. For purposes of 
identification according to the invention, an LCR subregion that does not 
include a chromatin opening domain will confer less than about 0.1% 
transcription on a linked transgene when carried in as a single copy 
construct, with respect to endogenous gene transcription. For example, as 
described below, the .beta.-globin chromatin opening domain confers an 
approximately 60-fold higher level of transcription on the .beta.-globin 
transgene than the .beta.-globin enhancer region alone confers on this 
transgene. Moreover, the .beta.-globin enhancer region (e.g., HS2) alone 
confers expression on a transgene in a non-reproducible manner. As used 
herein, "significantly higher" may refer to a level of expression that is 
about 5-fold or higher, for example, 10-fold, 25-fold, 50-fold, or as much 
as 80-100-fold higher. 
As used herein, "DNase I hypersensitive sites" are sites which are located 
in and around an expressed gene which are highly susceptible to cleavage 
by either DNase I or endogenous nucleases. As used herein, the term 
"hypersensitive" is inclusive of "super hypersensitive". A "DNase I 
hypersensitive site" refers to a region of DNA that is susceptible to 
DNase I at a concentration of at least 0.1 ug/ml at 37.degree. C., which 
susceptibility occurs prior in time to susceptibility of the remainder of 
DNA (i.e., non-hypersensitive DNA) that is cleaved in a time course of 
incubation. Other concentrations of DNase I which may distinguish 
hypersensitive DNA from non-hypersensitive sites include, for example, 1-5 
ug/ml, 10 ug/ml, or even higher concentrations such as 50-100 ug/ml DNase 
I. This variation in minimum DNase I concentration that distinguishes a 
hypersensitive site from non-hypersensitive DNA reflects the differing 
susceptibilities of different nuclear DNA preparations to nucleases. The 
presence of a "DNase I hypersensitive site" may be definitively determined 
by the appearance of a DNA fragment (i.e., a band) on a Southern blot due 
to preferential cutting of DNase I at a hypersensitive site. 
Further features and advantages of the invention will become more fully 
apparent in the following description of the embodiments and drawings 
thereof and from the appended claims.

DETAILED DESCRIPTION OF THE INVENTION 
The invention is illustrated by the following nonlimiting examples wherein 
the following materials and methods are employed. The entire disclosure of 
each of the literature references cited hereinafter are incorporated by 
reference herein. 
The invention is based on the discovery that certain functional properties 
of a locus control region may be physically and functionally separated. 
One such functional property is the ability of the LCR to transform the 
chromatin surrounding it into an open chromatin structure. The chromatin 
opening activity is essential in order to actuate transcription of an 
associated transgene regardless of the site of integration of the 
transgene in a host cell genome, but is in itself usually insufficient to 
give rise to physiological levels of transcription as the chromatin 
opening domains do not necessarily have enhancer activity. It is the 
identification and isolation of LCR chromatin opening activity that is the 
subject of the invention. The examples provided below enable 
identification and isolation of an LCR subregion containing a chromatin 
opening domain from any locus containing a tissue-specifically expressed 
gene that is under the control of Locus Control Region. 
Example I provides methods that are generally useful in carrying out the 
invention, and that were used to identify and characterize the 
.beta.-globin chromatin opening domain. Example II provides several LCR 
constructs that comprise fully functional LCRs. Example III teaches one of 
skill in the art how to identify and characterize an LCR subregion 
comprising a chromatin opening domain. The LCR subregion characterized in 
Example III is the 5'HS3 region. Example IV teaches one of skill in the 
art how to recognize an LCR subregion that does not contain a chromatin 
opening domain. Example V teaches one of skill in the art how to identify 
and characterize other chromatin opening domains of the invention in a 
reproducible and predictable manner. Example VI teaches one of skill in 
the art how to use constructs of the invention. Example VII teaches one of 
skill in the art how to utilize the invention for gene therapy involving 
blood disorders. Example VIII teaches one of skill in the art how to 
utilize the invention for gene therapy involving cells of the immune 
system and malignant cells. Example IX teaches one of skill in the art how 
to utilize a chromatin opening domain in combination with a heterologous 
enhancer. Example X teaches one of skill in the art how to utilize the 
invention to treat Gaucher's disease. Example XI teaches one of skill in 
the art how to utilize the invention to treat X-linked 
.gamma.-globulinemia. Example XII teaches one of skill in the act how to 
test for a chromatin opening domain using a reporter construct. 
EXAMPLE I 
Methods Useful in Carrying Out the Invention 
The following methods are routinely used in the invention. These methods 
are stated in terms of the detailed experiments performed herein for 
identification and isolation of the .beta.-globin HS3 chromatin opening 
domain. However, each method may be generalized by one of skill in the art 
for use in identifying and isolating other chromatin opening domains. 
1. Generation of Transgenic Mice 
Transgenic mice are generated by microinjection of about 0.25-0.50 ng/.mu.l 
purified DNA, as described (Ellis et al., Sem. Dev. Biol., 4:359-369, 
1993; Ellis et al., Eur. Mol. Biol. J. 12:127-134, 1993, both references 
of which are hereby incorporated), and screened by Southern blot 
hybridization and PCR on tail or fetal head DNA by standard procedures. Of 
the transgenic mice described below, 42 founder transgenic mice generated, 
2 died, 3 were infertile, 2 failed to transmit, 1 transmitted only an 
incomplete transgene, and 8 transmitted in a mosaic manner. Sequences of 
PCR primers useful in .beta.-globin locus fragment detection (see FIGS. 4 
and 8) include: 
1) 5'AAGCACAGCAATGCTGAGTCATG3' (SEQ ID NO: 1) 
2) 5'TCAATGGGGTAATCAGTGGTGTC3' (SEQ ID NO: 2) 
3) 5'GGGTGGGAGAATCAGGAAACTAT3' (SEQ ID NO: 3) 
4) 5'GTCTTAGCCAGTTCCTTACAGCT3 (SEQ ID NO: 4) 
5) 5'TGTCACATTCTGTCTCAGGCATC3' (SEQ ID NO: 5) 
6) 5'TGCCAGATGTGTCTATCAGAGGT3' (SEQ ID NO: 6) 
7) 5.degree. CATGGTTTGACTGTCCTGTGAGC3' (SEQ ID NO: 7) 
8) 5'GGTGGTTGATGGTAACACTATGC3' (SEQ ID NO: 8) 
2. DNA Analysis 
Southern transfer and hybridization are performed by standard procedures. 
Copy-number determination is performed using a Molecular Dynamic 
PhosphorImager and adjusted for loading differences or the presence of 
non-intact transgenes (observed after digestion with BspH1 or other 
diagnostic restriction enzymes). In order to determine definitively that a 
transgene is in single copy, it was found to be essential to examine end 
fragments in both directions. Digestions described below were therefore 
performed with Eco RI for the 5' end fragment and Bam HI for the 3' end 
fragment. Southern blots were probed with the .beta.ivs2 probe. 
3. RNA Analysis 
Fetal liver RNA (13.5-15.5 day) was extracted, 1 .mu.g was hybridized to 
kinased double-stranded DNA probes, digested with 75 U S1 nuclease, and 
run on a 6% sequencing gel as described (Ellis et al., Sem. Dev. Biol., 
4:359-369, 1993; Ellis et al., Eur. Mol. Biol. J. 12:127-134, 1993). Probe 
excess was demonstrated by including a sample containing 3 .mu.g fetal 
liver RNA. Specific activities of human .beta.-globin (H.beta.) relative 
to the mouse .beta.major (.beta.maj) probe was 2:1. The protected 160 nt 
H.beta. and 95 nt .beta.maj bands were quantified on a Molecular Dynamics 
PhosphorImager and the % expression levels calculated according to the 
formula (H.beta./2.beta.maj).times.100 to account for the specific 
activity differences. % Expression per copy was calculated as (2 .beta.maj 
genes/number H.beta. transgenes).times.% expression. 
4. Vectors 
Recombinant retroviral vectors as well as other DNA transfer schemes can be 
used in practice of the present invention. A recombinant viral vector of 
the invention will include DNA of at least a portion of a retroviral 
genome which portion is capable of infecting the target cells and a 
functional gene operatively linked thereto. As used herein, "functional" 
or "expressible" gene means a gene encoding a protein having a biological 
effect. By "infection" is generally meant the process by which a virus 
transfers genetic material to its host or target cell. Preferably, the 
retrovirus used in the construction of a vector of the invention is also 
rendered replication-defective to remove the effects of viral replication 
on the target cells. In such cases, the replication-defective viral genome 
can be packaged by a helper virus in accordance with conventional 
techniques. Generally, any retrovirus meeting the above criteria of 
infectiousness and capabilities of functional gene transfer can be 
employed in the practice of the invention. 
Suitable retroviruses for practice of the invention include but are not 
limited to, for example, adenoviruses, adeno-associated virus and SV40 
virus; suitable retroviral vectors include but are not limited to pLJ, 
pZip, pWe and pEM, well known to those skilled in the art; suitable 
packaging virus lines for replication-defective retroviruses include, for 
example, .psi.Crip, .psi.Cre, .psi.2 and .psi.Am. 
It will be appreciated that when viral vector schemes are employed for gene 
transfer according to the invention, the use of an attenuated or a 
virulent virus also may be desirable. 
The genetic material to be recombined with the retroviral vector or 
transferred through other methods of the invention is preferably provided 
through conventional cloning methods, i.e., cDNA, through overlapping 
sequences or any other suitable method yielding the desired clone. 
An Adeno-Associated Virus (AAV) vector is representative of retroviral 
vectors useful according to the invention. AAV is a defective human 
parvovirus with no known pathogenicity. AAV contains a linear 
single-stranded DNA of 4.7 kb in length. The AAV genome carries two sets 
of functional genes: the rep genes, which encode proteins necessary for 
viral replication, and the structural capsid protein genes. AAV DNA also 
includes a 145 bp Inverted Terminal Repeat (ITR) at each end, between 
which lie the two sets of genes arranged in three major transcription 
units. Two transcription units overlap and encode a family of four related 
rep proteins, and the third encodes the virus capsid protein (Samulski, 
1993, Curr. Opin. Genet. Devel. 3:74). 
Knowledge of the AAV life-cycle has been applied to develop AAV based 
vectors and vector packaging cell lines for stably transducing mammalian 
cell lines. The principles of these systems are similar to those on which 
retroviral vectors are based (Muzyczka, 1992, Curr. Top. Microbiol. 
Immunol., 158:98). A plasmid harboring an expression cassette no greater 
than 4.7 kb in length and located between two AAV ITRs, is co-transfected 
with a plasmid encoding expressible AAV Rep and Capsid genes into a helper 
cell line productively infected with adenovirus. Culture supernatant from 
these transfectants is highly enriched for recombinant AAV virions 
containing single stranded DNA which encodes the expression cassette 
flanked by ITRs. When such virions are used in gene transfer experiments, 
stable transductants can be derived, and transduction is more efficient in 
cells passing through S-phase (Russell et al., 1994, Proc. Nat. Acad. 
Sci., 91:8915-8919), but integration of their recombinant genomes into 
host DNA appears to be random. To date, a number of AAV-based vectors 
packaged in this way have been used to stably transduce human 
T-lymphocytes, fibroblasts, nasal polyp, erythroid, and haemopoietic stem 
cells for gene therapy applications (Philip et al., 1994, Mol. Cell. 
Biol., 14:2411-2418; Russell et al., 1994, Proc. Nat. Acad. Sci., 
91:8915-8919; Flotte et al., 1993, Proc. Nat. Acad. Sci., 90:10613-10617; 
Walsh et al., 1994, Proc. Nat. Acad. Sci., 89:7257-7261; Miller et al., 
1994, Proc. Nat. Acad. Sci., 91:10183-10187). One of these reports 
describes the incorporation, into a helper-line-packaged AAV vector, of 
the .beta.-globin Locus Control Region driving expression of the HbF 
.gamma. chain gene, for possible use in gene therapy of Sickle Cell 
Disease (Miller et al., 1994, Proc. Nat. Acad. Sci., supra). See also PCT 
publication WO91/18088, by Chatterjee et al., for additional AAV-based 
eucaryotic vectors. 
One advantage of using retroviral vectors, and in particular AAV vectors, 
is that foreign DNA introduced into mammalian cells remains attached to 
the AAV proviral genome and comprises a stable and heritable portion of 
the mammalian cell genome. Retroviral-mediated gene transduction into a 
cell according to the invention is optimized using a replication defective 
retrovirus. 
Accordingly, a chromatin opening domain, as described herein, may be 
combined with a functional gene in an AAV vector. The vector is then 
administered to a patient, using a vector delivery system as described 
herein, to treat a disease which is associated with absence or mutation of 
the protein encoded by the functional gene. Administration of the vector 
will result in expression of the functional gene, in the cell into which 
the vector is introduced, in a position independent manner, the latter 
property being conferred by the chromatin opening domain. 
5. Lipsomal Gene Transfer 
Liposomes have been used for non-viral delivery of many substances, 
including nucleic acids, viral particles, and drugs. A number of reviews 
have described studies of liposome production methodology and properties, 
their use as carriers for therapeutic agents and their interaction with a 
variety of cell types. See, for example, "Liposomes as Drug Carriers," 
Wiley and Sons, New York (1988), and "Liposomes from Biophysics to 
Therapeutics," Marcel Dekker, New York (1987). Several methods have been 
used for liposomal delivery of DNA into cells, including poly-L-lysine 
conjugated lipids (Zhou et al., Biochim. Biophys. Acta. 1065:8-14, 1991), 
pH sensitive immunoliposomes (Gregoriadis, G., Liposome Technology, Vol I, 
II, III, CRC, 1993), and cationic liposomes (Felgner et al., Proc. Natl. 
Acad. Sci., USA, 84:7413-7417, 1987). Positively charged liposomes have 
been used for transfer of heterologous genes into eukaryotic cells 
(Felgner et al., 1987, Proc. Nat. Aca. Sci. 84:7413; Rose et al., 1991, 
BioTechniques 10:520). Cationic liposomes spontaneously complex with 
plasmid DNA or RNA in solution and facilitate fusion of the complex with 
cells in culture, resulting in delivery of nucleic acid to the cell. 
Philip et al. 1994, Mol. and Cell. Biol. 14:2411, report the use of 
cationic liposomes to facilitate adeno-associated virus (AAV) plasmid 
transfection of primary T lymphocytes and cultured tumor cells. 
Delivery of an agent using liposomes allows for noninvasive treatment of 
diseases. Targeting of an organ or tissue type may be made more efficient 
using immunoliposomes, i.e., liposomes which are conjugated to an antibody 
specific for an organ-specific or tissue-specific antigen. Thus, one 
approach to targeted DNA delivery is the use of loaded liposomes that have 
been made target-specific by incorporation of specific antibodies on the 
liposome surface. Immunoliposome-associated reagents have been reported to 
result in less than optimal accumulation at target sites, possible due to 
sequestration by the reticuloendothelial system, primarily by the liver 
and spleen. Torchilin et al. (FASEB Journal 6:2716, 1992) report on 
enhancement of circulation times using polyethylene glycol-coated 
immunoliposomes. The invention will therefor encompass liposomal delivery 
of a DNA construct using such modified liposomes. 
Liposomes are composed of a bilayer lipid matrix that wraps around an 
aqueous volume, thus isolating it from the external medium. The central 
aqueous core may vary in diameter from 20 nm to as much as 2-3 
micrometers. The term "liposome", as used herein, is also intended to 
encompass liposomes which are composed of several (e.g., 2-3) concentric 
bilayers which define several individual aqueous compartments. 
Thermodynamically, liposomes have minimum free energy as long as the 
density of the phospholipids in each monolayer of the bilayer structure is 
the same. 
Liposomes useful in the invention are composed of phospholipid molecules. A 
phospholipid molecule has a polar head group and two nonpolar, hydrophobic 
fatty acyl chains. In an aqueous environment, the most energetically 
stable form for phospholipids is within structures that allow the fatty 
acyl chains to avoid contact with water. A lipid bilayer is one such 
structure. Many phospholipids, when dispersed in water, spontaneously form 
lipid bilayer structures. Lipid bilayer structures useful in the invention 
are preferably circular structures. 
The polar head group of the phospholipid molecule may include choline, 
e.g., lecithins (phosphatidylcholines) and sphingomyelins. Such molecules 
may also include amino groups, e.g., phosphatidylserine and phosphatidyl 
ethanolamine. Other polar head groups may include phosphatidylglycerol, 
phosphatidylinositol and cardiolipin. 
An immunoliposome will include a liposome component, as described above, 
conjugated via its polar head group to the carboxy terminus of an 
immunoglobulin molecule. Fusion of an immunoliposome with the cell 
membrane will occur because most cell membranes are composed of protein 
and phospholipid bilayers. Immunoglobulins will be used for targeting 
liposomes to selected cells. 
a) Preparation of Liposomes and Immunoliposomes 
Liposomes and immunoliposomes may be prepared according to a variety of 
techniques, e.g., detergent dialysis or the formation of a water-in-oil 
emulsion, slow swelling in nonelectrolytes, dehydration followed by 
rehydration, dilution or dialysis of lipids in the presence of chaotropic 
ions, and mechanical preparation techniques such as freeze-thaw cycling. 
Removal of detergent molecules from aqueous dispersions of 
phospholipid/detergent mixed micelles represents one way of producing 
liposomes (see J. Biol Chem. 246:5477 (1971) herein incorporated by 
reference). As the detergent is removed, the micelles become progressively 
richer in phospholipid and finally coalesce to form closed, single bilayer 
vesicles. Detergents commonly used for this purpose include bile salts and 
octylglycoside. Because this method does not involve the use of organic 
solvents and sonication, it is particularly useful for entrapping 
macromolecules, such as nucleic acids, which are sensitive to the presence 
of organic solvents or are structurally altered by sonication. 
Another method of preparing liposomes is the reverse phase evaporation 
method detailed in U.S. Pat. No. 4,235,871, which is incorporated herein 
by reference. Liposomes prepared by this method have a typical average 
size of about 2-4 microns and are predominantly oligolamellar, that is, 
contain one or a few lipid bilayer shells. 
Liposomes may also be prepared via hydration in the presence of a solvent. 
Multi-lamellar vesicles (MLVs) with high encapsulation efficiency can be 
prepared by hydrating the lipids in the presence of an organic solvent. 
The two phases are emulsified by vigorous mixing (vortexing) and then the 
organic phase removed by passing a stream of nitrogen gas over the 
emulsion. As the solvent evaporates, liposomes form in the aqueous phase. 
Mechanical preparation methods, e.g., shaking by hand, sonication, French 
pressure freeze-drying, membrane extrusion, freeze-thawing, changing pH, 
calcium inducing, and micro emulsion techniques, have been used for the 
preparation of liposomes. In essence, a mixture of vesicle-forming lipids 
in a volatile organic solvent is deposited on the surface of a round 
bottomed flask, and the solvent is removed by rotary evaporation under 
reduced pressure. Vesicles ranging in size from one-tenth to tens of 
microns form spontaneously when an excess volume of aqueous buffer is 
added with agitation to the dry lipid. 
Methods for controlling the size of liposomes are various and include 
extrusion and homogenization. One effective sizing method involves 
extruding an aqueous suspension of the liposomes through a series of 
polycarbonate membranes having a selected uniform pore size in the range 
of 0.2-0.6 micron, typically 0.1-0.2 micron. The pore size of the membrane 
corresponds roughly to the largest sizes of liposomes produced by 
extrusion through that membrane, particularly where the preparation is 
extruded two or more times through the same membrane. Extrusion of 
liposomes can also be performed through an asymmetric ceramic filter, as 
taught in U.S. Pat. No. 4,737,323, herein incorporated by reference. Other 
methods of reducing particle size include application of high pressures to 
the liposomes, as in a French Press, and homogenization of the liposomes. 
Antibodies have been developed to cell-surface antigens for targeting of 
numerous cell types, including but not limited to malignant cells. 
Techniques are known for conjugating such antibodies to pharmacologically 
active agents or to labels to permit diagnosis, localization, and therapy 
directed toward such tumors. 
Liposome targeting based on antibody/antigen recognition has been utilized 
in the prior art in the development of targeted delivery systems for 
delivery of various bioactive agents to a target site. Antibody-directed 
liposomes, or immunoliposomes, are used for this purpose. Antibody 
molecules are predominantly hydrophilic compounds with no affinity for the 
hydrophobic liposome membrane. Immunoliposomes can be used to deliver 
hundreds or more units of intraliposomal contents into an individual 
target cell. Immunoliposomes administered according to the invention are 
administered intravenously, intraperitoneally or directly to the target 
tissue or organ, at a dosage that is appropriate for the amount of 
biological agent or genetic material that is encapsulated by the liposome. 
Immunoliposome dosage will therefore vary from about 5 mg/kg body weight 
to about 1 gm/kg body weight, and may be in the range of 100 mg-500 mg/kg 
body weight. 
As used herein, an immunoliposome comprises a liposome conjugated to an 
immunoglobulin molecule. Generally and as used herein, a 
liposome/immunoglobulin conjugate comprises an immunoglobulin molecule 
linked via direct or indirect means and either covalently or noncovalently 
to the phospholipid molecule. Generally, about 1 in every 200 phospholipid 
molecules of the liposome will be linked to an antibody molecule, with an 
acceptable range being 1 in every 20-20,000 phospholipid molecules of the 
liposome. 
Where enhancement of specificity of the immunoliposome for the target site 
is desired, the immunoliposome may include antibodies of several different 
specificities, each cognate antigen being found at the target site. Such 
multiple specificity may also be conferred using bifunctional or 
trifunctional antibodies (see, e.g., U.S. Pat. No. 5,237,743, hereby 
incorporated by reference). 
Methods are known in the prior art for preparing immunoliposomes. 
Immunoliposomes are prepared, for example, by adsorption of proteins 
(e.g., immunoglobulin) on the liposomal surface; incorporation of native 
protein into the liposome membrane during its formation (e.g., by 
ultrasonication, detergent dialysis or reverse phase evaporation); 
covalent binding (direct or via a spacer group) of a protein to reactive 
compounds incorporated into the liposomes membrane; noncovalent 
hydrophobic binding of modified proteins during liposome formation or by 
the incubation with preformed liposomes); and indirect binding, including 
covalent binding of immunoglobulin protein via a polymer to the liposome 
(see Torchilin, V. P. CRC Critical reviews in Therapeutic Drug Carrier 
Systems, vol. 2(1), hereby incorporated by reference). 
Immunoliposomes may be prepared according to the following procedure. 
1. Covalent Coupling of Antibody with NGPE. 
0.6 mg N-Glutaryl phosphatidylethanolamine (NGPE) was dissolved in 0.5 cc 
2-[N-morpholino]ethanesulfonic acid hemisodium salt (MES) buffer (in 0.016 
M octylglucoside in 50 mM MES). After the addition of 4.8 mg 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 6 mg 
N-hydroxysulfosuccinimide (HSSI), the resulting mixture is incubated at 
room temperature for 5 min. The antibody solution (containing antimyosin 
antibody 2G42D7, described below, or other antibody) is then added (0.36 
mg/ml). The pH of the mixture is then adjusted to 8.0 with 1M NaOH. The 
reaction mixture is incubated at 4.degree. C. for 8-12 hour with mixing. 
The resulting NGPE-antibody conjugate is then dialyzed overnight against 
PBS, pH 7.4 to remove octylglucoside and other excess reagents. 
2. Preparation of Immunoliposomes by Detergent Dialysis. 
Liposomes are prepared from a mixture of egg phosphatidylcholine (PC) and 
cholesterol (Ch) in chloroform in the molar ratio 1:1. The lipid mixture 
(30 mg PC/17.96 mg Ch) is dried with argon, then vacuum dried for 2 hour 
and resuspended in 4 cc phosphate buffered saline (PBS) containing 0.016 M 
octylglucoside with brief ultrasonication. The solution of NGPE-modified 
antibody (0.7 mg/ml) is added to solubilized lipids. The mixture is 
dialyzed overnight against PBS (pH 7.4) to remove detergent. The resulting 
liposomes are extruded through a Nucleopore filter (0.6, 0.4, and 0.2 
.mu.m). The same method is used for preparation of liposomes without 
NGPE-antibody solution. 
b) Loading of Immunoliposomes 
Loading of compounds into liposomes may be achieved by one or more of a 
variety of active and passive methods. Passive loading by entrapment is 
employed where relatively low concentrations of the DNA construct is 
desired. Loading of high concentrations of DNA in liposomes may require 
active loading methods, e.g., as described in U.S. Pat. No. 5,129,549, 
herein incorporated by reference, in which a chemical gradient is created 
across the liposome membrane that results in trapping of the DNA in the 
internal aqueous phase of the liposome. 
Liposome/DNA formulations are characterized by measurements of particle 
size, lipid concentration, and pH by standard methods as described above. 
DNA incorporation into the composition may be determined by inclusion of 
radiolabeled tracer in the composition. The amount of liposome-entrapped 
DNA is then determined by gel permeation chromatography using BioRad A-15M 
resin. The liposomal DNA fraction is calculated from the amount of 
radiolabel present in the void volume of the column, and the percentage of 
liposomal DNA from the ratio of label eluting in the void volume to the 
remaining label eluting from the column. 
7. Preparation of Antibodies 
Immunoglobulin molecules useful in the invention include whole antibody, or 
any antibody fragment, for example, a F(ab')2, Fab, and/or an Fv fragment 
of an antibody molecule. In addition, any variable region specificity of 
an antibody molecule is useful according to the invention. 
A F(ab')2 fragment is that portion of an antibody molecule which contains 
the complete antigen-combining site, consisting of two light chains and 
part of each heavy chain, and is produced by enzymatic digestion, e.g., 
using pepsin, such that the heavy chain disulfide bonds remain intact in 
the F(ab')2 fragment. A Fab fragment consists of a single light chain and 
a part of a heavy chain disulfide bonded together. Fab is produced by 
enzymatic digestion, e.g., using papain, such that about one-half the 
F(ab')2 antigen binding fragment is generated. An Fc fragment is that 
portion of an antibody that is responsible for binding to antibody 
receptors on cells and the Clq component of complement. The Fc fragment is 
the portion of the antibody molecule that remains after papain digestion. 
An Fv fragment is that portion of an antibody consisting of the variable 
region of a Fab fragment. 
Antibodies useful in the invention may be obtained through conventional 
polyclonal or monoclonal antibody preparation techniques. Antigen may be 
obtained from cells of the species toward which the antibodies are to be 
directed. Such species are preferably vertebrate, more preferably 
mammalian, and most preferably human. For antibodies directed toward human 
intracellular antigens, immortal cell lines represent a convenient source 
of such antigen. 
To generate monoclonal antibodies, murine spleen cells from immunized 
animals are fused with an appropriate myeloma cell line. Fused cells are 
cultured in selective growth medium to establish hybridoma colonies, each 
colony secreting an antibody of interest. Culture supernatants from each 
colony are then tested for antibody specificity. Positive cultures are 
identified and expanded. See Kohler et al., Nature 256:495 (1975), hereby 
incorporated by reference. 
8. Delivery of Gene via DNA-Protein Complexes 
Transfer of a DNA construct according to the invention can be accomplished 
through many means, including but not limited to transfection using 
calcium phosphate coprecipitation, fusion of the target cell with 
liposomes, erythrocyte ghosts or spheroplasts carrying DNA, plasmid and 
viral vector-mediated transfer, and DNA protein complex-mediated gene 
transfer such as receptor-mediated gene transfer. 
Receptor-mediated gene transfer is dependent upon the presence of suitable 
ligands on the surfaces of cells which will allow specific targeting to 
the desired cell type followed by internalization of the complex and 
expression of the DNA. One form of receptor-mediated gene transfer is 
wherein a DNA vector is conjugated to antibodies which target with a high 
degree of specificity cell-surface antigens (Wong and Huang, 1987, Proc. 
Nat. Aca. Sci. 84:7851; Roux et al., 1989, Proc. Nat. Aca. Sci. 86::9079; 
Trubetskoy et al., 1992, Bioconjugate Chem. 3:323; and Hirsch et al., 
1993, Transplant Proceedings 25:138). Nucleic acid may be attached to 
antibody molecules using polylysine (Wagner et al., 1990, Proc. Nat. Aca. 
Sci. 87:3410; Wagner et al., 1991, Proc. Nat. Aca. Sci. 89:7934) or via 
liposomes, as described below. 
Increased expression of DNA derived from ligand-DNA complexes taken up by 
cells via an endosomal route has been achieved through the inclusion of 
endosomal disruption agents, such as influenza virua hemagglutinin 
fusogenic peptides, either in the targeting complex or in the medium 
surrounding the target cell. An enhanced transfection protocol which 
allows for targeted delivery and uptake of nucleic acid vectors to 
specific cells at high efficiency, preferably in the absence of 
purification of the cells from a mixed cell population is described in 
PCT/GB94/01835. 
Targeted gene delivery is also achieved according to the invention using a 
DNA-protein complex. Such DNA-protein complexes include DNA complexed with 
a ligand that interacts with a target cell surface receptor. Cell surface 
receptors are thus utilized as naturally existing entry mechanisms for the 
specific delivery of genes to selected mammalian cells. It is known that 
most, if not all, mammalian cells possess cell surface binding sites or 
receptors that recognize, bind and internalize specific biological 
molecules, i.e., ligands. These molecules, once recognized and bound by 
the receptors, can be internalized within the target cells within 
membrane-limited vesicles via receptor-mediated endocytosis. Examples of 
such ligands include but are not limited to proteins having functional 
groups that are exposed sufficiently to be recognized by the cell 
receptors. The particular proteins used will vary with the target cell. 
Typically, glycoproteins having exposed terminal carbohydrate groups are 
used although other ligands such as antibodies or polypeptide hormones, 
also may be employed. Using this technique the phototoxic protein psoralen 
has been conjugated to insulin and internalized by the insulin receptor 
endocytotic pathway (Gasparro, Bio-chem. Biophys. Res. Comm. 141(2), pp. 
502-509, Dec. 15, 1986); the hepatocyte specific receptor for galactose 
terminal asialoglycoproteins has been utilized for the hepatocyte-specific 
transmembrane delivery of asialoorosomucoid-poly-L-lysine non-covalently 
complexed to a DNA plasmid (Wu, G. Y., J. Biol. Chem., 262(10), pp. 
4429-4432, 1987); the cell receptor for epidermal growth factor has been 
utilized to deliver polynucleotides covalently linked to EGF to the cell 
interior (Myers, European Patent Application 86810614.7, published Jun. 6, 
1988); the intestinally situated cellular receptor for the organometallic 
vitamin B.sub.12 -intrinsic factor complex ahs been used to mediate 
delivery to the circulatory system of a vertebrate host a drug, hormone, 
bioactive peptide or immunogen complexed with vitamin B.sub.12 and 
delivered to the intestine through oral administration (Russel-Jones et 
al., European patent Application 86307849.9, published Apr. 29, 1987); the 
mannose-6-phosphate receptor has been used to deliver low density 
lipoprotiens to cells (Murray, G. J. and Neville, D. M., Jr., J. Bio. 
Chem. Vol 225 (24), pp. 1194-11948, 1980); the cholera toxin binding 
subunit receptor has been used to deliver insulin to cells lacking insulin 
receptors (Roth and Maddox, J. Cell. Phys. Vol. 115, p. 151, 1983); and 
the human chorionic gonadotropin receptor has been employed to deliver a 
ricin a-chain coupled to HCG to cells with the appropriate HCG receptor in 
order to kill the cells (Oeltmann and Heath, J. Biol. Chem, vol 254, p. 
1028 (1979)). Ligands selected from biotin, biotin analogs and biotin 
receptor-binding ligands, and/or folic acid, folate analogs and folate 
receptor-binding ligands to initiate receptor mediated transmembrane 
transprot of the ligand complex, as described in U.S. Pat. No. 5,108,921. 
Generally, a ligand is chemically conjugated by covalent, ionic or hydrogen 
bonding to the nucleic acid. A ligand for a cell surface receptor may be 
conjugated to a polycation such as polylysine with ethylidene diamino 
carbodiimide as described in U.S. Pat. No. 5,166,320. DNA may be attached 
to an appropriate ligand in such a way that the combination thereof or 
complex remains soluble, is recognized by the receptor and is internalized 
by the cell. The DNA is carried along with the ligand into the cell, and 
is then expresssed in the cell. The protein conjugate is complexed to DNA 
of a transfection vector by mixing equal mass quantities of protein 
conjugate and DNA in 0.25 molar sodium chloride. The DNA/protein complex 
is taken up by cells and the gene is expressed. 
Delivery of the foreign DNA into the target cell may also be achieved via 
the DNA construct's association with an endosomal disruption agent, such 
as the influenza hemagglutinin fusogenic peptide. The fusogenic peptide of 
the HA molecule is a modified form of HA which retains two important 
functions of HA. It allows for fusion of the targeted DNA/ligand complex 
to the cell membrane, but without the host cell sialic acid-binding 
specificity of the natural molecule. Instead, host cell binding 
specificity is conferred by the ligand/receptor interaction. The modified 
HA fusogenic peptide also retains the HA function of endosomal uptake, 
thus allowing for uptake of the complex into the host cell via membrane 
fusion, and the endosomal escape function of HA, which allows for escape 
of the enveloped DNA from the endosomal/lysosomal destruction pathway. 
Thus, the invention encompasses a composition of matter for targeted 
delivery of DNA to a target cell. The composition comprises (a) a DNA 
construct containing an expressible gene encoding a protein of interest 
and a chromatin opening domain which domain confers position independent 
expression on the expressible gene; and (b) a ligand capable of binding to 
a target cell. Thus, the DNA construct will be complexed with the ligand 
such that when the ligand targets its cognate receptor on the target cell, 
the construct is physically carried along. 
As described inter alia, the composition may further include (c) an 
endosomal disruption agent, for example an influenza hemagglutinin (HA) 
fusogenic peptide such as the HA amino acid sequence 
GLFGAIAGFIGAGTGGMIAGGGC (SEQ ID NO:9). 
The ligand may include an antibody that is specific for a surface antigen 
of the target cell. Such antibodies may include but are not limited to 
antibodies to any immune cell surface antigen, e.g., as are present on 
immune precursor cells, B-cells, T-cells, andmacrophages, i.e., CD19, 
CD20, CD21, CD22, CD38, CD72, MHCII, etc. 
9. Target Cells 
The cells targeted for transduction or gene transfer in accordance with the 
invention include any cells to which the delivery of the functional gene 
is desired, for example, immune cells such as T-cells, B-cells, 
macrophages, hematopoietic cells, and dendritic cells. Cells or cell 
populations can be treated in accordance with the invention in vivo or in 
vitro. Using established technologies, stem cells may be used in DNA 
transfection after enrichment procedures (see, for example, European 
Patent Applications 0 455 482 and 0 451 611, which disclose methods for 
separating stem cells from a population of hematopoietic cells). 
Alternatively, hematopoietic cells and stem cells may be made susceptible 
to DNA uptake using the method described in PCT/GB94/01835 which allows 
for targeted delivery and uptake of nucleic acid vectors to specific cells 
at high efficiency, preferably in the absence of purification of the cells 
from a mixed cell population. DNA may be transferred into B-cells or pre-B 
cells using published procedures; see, for example, Martensson et al., 
Eur. Jour. Immunol., 1987, 17:1499; Okabe et al., Eur. Jour. Immunol., 
1992, 22:37; and Banerji et al., 1983, Cell 33:729). DNA is transferred 
into T-cells or cell lines via the procedure described in Philip et al., 
1994, Mol. and Cell. Biol. 14:2411. DNA is transferred into macrophages as 
described in Immunol. and Cell Biol. 71:75, 1993. 
In in vivo treatments, vectors of the invention can be administered to the 
patient, preferably in a biologically compatible solution or a 
pharmaceutically acceptable delivery vehicle, by ingestion, injection, 
inhalation or any number of other methods. The dosages administered will 
vary from patient to patient and will be determined by the level of 
enhancement of function of the transferred genetic material balanced 
against any risk or deleterious side effects. Monitoring levels of 
transduction, gene expression and/or the presence or levels of normal 
encoded protein will assist in selecting and adjusting the dosages 
administered. In vitro transduction is also contemplated within the 
present invention. Cell populations with defective genes can be removed 
from the patient or otherwise provided, transduced with a normal gene in 
accordance with the invention, the reintroduced into the patient. 
EXAMPLE II 
LCR Constructs That Comprise Fully Functional LCRs 
An LCR construct that comprises a fully functional LCR is identified and 
described in this Example. This construct functions at single copy and is 
of a reduced size compared to the significantly larger complete LCR 
construct known in the prior art (FIG. 1A-B). The microlocus LCR construct 
contains all four hypersensitive sites on a 6.5 kbp cassette upstream of 
the .beta.-globin gene (FIG. 1B), it includes a 2.1 kb XbaI fragment 
encompassing DNase site HS1, a 1.9 kb HindIII fragment encompassing HS2, a 
1.5 kb Asp718-SalI fragment encompassing HS3, a 1.1 kb partial SacI 
fragment encompassing HS4, and the 4.9 kb BglII fragment containing the 
.beta.-globin gene, as described in U.S. Ser. No. 07/920,536 and WO 
89/01517. The complete DNA sequence of 16 kb of DNA 5' to the human 
.beta.-globin gene is described in Li et al., 1985, Jour. Biol. Chem. 
241:28;14901, hereby incorporated by reference. The microlocus construct 
was investigated with respect to whether it retained fully functional LCR 
activity. Eight .mu.D founder transgenic mice were generated containing 
the microlocus construct; five lines were established with a range of copy 
numbers from 1 to 9 (FIGS. 1C and D). S1 analysis of fetal liver RNA 
showed that both single-copy .mu.D lines expressed human .beta.-globin at 
an average of 47% the level produced by the two mouse .beta.major genes or 
94% per copy (FIG. 2). In this case, the positive control "line 72" was 
calculated to contain 53% human .beta.-globin or 106% per copy. Line 72 
contains a single-copy of the entire human .beta.-globin locus (Stouboulis 
et al., 1992, supra). These data demonstrate that the 6.5 kbp microlocus 
cassette fully activates single-copy .beta.-globin transgene expression in 
a reproducible manner and therefore falls within the definition of a fully 
functional LCR. The .beta.-globin microlocus LCR, when carried in single 
copy in a transgenic mouse line, is referred to as line .mu.D14. The 
microlocus contains a chromatin opening domain that directs reproducible 
expression of independent single-copy transgenes, in addition to several 
enhancer elements, the combinations of elements of which provide for a 
physiological level of expression of the .beta.-globin gene. 
EXAMPLE III 
How to Identify and Characterize an LCR Subregion Comprising a Chromatin 
Opening Domain 
A) Identification of the .beta.-globin Chromatin Opening Domain 
FIG. 3A is a schematic illustration of the .beta.-globin locus, showing the 
four DNase I hypersensitive sites that constitute the LCR. FIG. 3B 
illustrates three constructs tested herein in single copy transgenic 
animals for chromatin opening domain activity; i.e., each of the HS2, HS3, 
and HS4 domains operatively associated with the human .beta.-globin gene. 
FIG. 3C illustrates the 1.9 kbp HS3 domain containing nuclear factor 
binding sites 1-6 (FP1-6) (not drawn to scale). In Example III, an LCR 
subregion comprising a chromatin opening domain is identified and defined. 
This subregion, identified and defined within the .beta.-globin LCR, is 
the 5'HS3 domain. The results showed that the chromatin opening domain 
activity within 5'HS3 does not lie solely within the portion of the region 
of 5'HS3 known as the core region, i.e., corresponding to footprints 1-3 
(see FIG. 3C). Nor is chromatin opening domain activity found within 
either of the 1.5 kbp 5'HS2 or 1.1 kbp 5'HS4 fragments, each of which 
contains a classical enhancer element. The HS2 and HS4 fragments are 
described in detail in PCT publication WO 89/01517, hereby incorporated by 
reference. HS2 is conveniently bound by HindIII sites to give a 1.5 kbp 
fragment; HS4 is bound by SacI sites to give a 1.1 kb fragment. In order 
to assess whether reproducible single-copy transgene expression activity 
requires all four hypersensitive sites in a microlocus arrangement or can 
be found within a specific smaller domain, the "C" construct (FIG. 4) 
which contains a .beta.-globin gene regulated by the 1.9 kbp 5'HS3 
fragment, was tested in single-copy in transgenic mouse lines. 
Six founder mice were generated bearing the C construct and bred to 
wild-type mice to obtain nonmosaic F.sub.1 fetuses representing 6 
independent mouse lines (FIG. 4). FIG. 4 is a map of the C gene construct, 
and provides ratios of transgenic/total mice generated for F.sub.0 or 
F.sub.1 generations. In FIG. 4, transgenic mice were generated using 0.25 
ng/.mu.l DNA as described (Kollias et al., 1986, supra; Ellis et al., 
1993, supra; Ellis et al., 1993, supra). Screening of tail DNA by Southern 
blot analysis and PCR were by standard methods. 
Five of the lines were shown to be single-copy as determined by the 
presence of unique 3' end fragments in Southern blot analysis of fetal 
head DNA digested with BamH1 and hybridized with a probe specific for 
human .beta.-globin (FIG. 5). The copy number of these lines was verified 
by Southern blot analysis of 5' end fragments in EcoR1 digested DNA, and 
in all cases transgene intactness was shown by Southern blot analyses on 
Stu1-EcoR1 or Pst1 digested DNA (data not shown). Transgene intactness was 
further confirmed by PCR analysis of the .beta.-globin 3' enhancer element 
using the primers whose locations in the construct are shown schematically 
in FIG. 4 (data not shown). 
S1 analysis was performed on 13.5 day fetal liver RNA to examine the 
expression status of the single-copy transgenes relative to the endogenous 
mouse 3-globin major genes (FIG. 6). In FIG. 6, 13.5 day fetal liver RNA 
was extracted and subjected to S1 nuclease analysis as described (Ellis et 
al., 1993, supra; Ellis et al., 1993, supra; Antoniou et al., 1988, 
supra). Specific activities of human .beta.-globin (H.beta.) relative to 
the mouse Bmajor (.beta.maj) probe was 2:1. The protected bands were 
quantified on a Molecular Dynamics PhosphorImager and the % expression 
levels calculated according to the formula (H.beta./2.beta.maj).times.100 
to account for the specific activity differences. % Expression per copy 
was calculated as (2 .beta.maj genes/number H.beta. transgenes X % 
expression. As positive controls for expression, we used F.sub.1 fetal 
liver RNA from Line 72 and Line .mu.D14. Line 72 (complete .beta.-globin 
LCR) and .mu.D14 (.beta.-globin microlocus) fetal liver expressed human 
.beta.-globin mRNA at approximately 47% and 45% of the level produced by 
the two mouse .beta.major genes, or 94% and 90% per copy respectively. As 
a negative control, we used nontransgenic (ntg) fetal liver RNA. In 
contrast to the full levels of expression produced by complete LCR 
constructs, .beta.-globin transgenes alone express at less than 1% per 
copy (Ryan et al., 1989, supra). 
Note that the B construct (FIG. 8), i.e., which contains a single-copy 
.beta.-globin transgene regulated by the 1.5 kbp 5'HS2 fragment, does not 
express a detectable level of human .beta.-globin, i.e., less than 0.1% 
per copy. This result demonstrates that the 5'HS2 enhancer element is 
suppressed by surrounding closed chromatin and hence is not an LCR element 
that possesses chromatin domain opening activity that functions in 
single-copy. However, the C lines containing the 1.9 kbp 5'HS3 fragment 
expressed significant human .beta.-globin levels with a mean average of 
26% per copy (range of 6-38% per copy) in all five single-copy lines. 
These data indicate that 5'HS3 is an LCR element that contains a chromatin 
opening domain which over-rides the suppressive effects of surrounding 
closed chromatin and reproducibly activates significant expression from 
independent integration sites. 
To investigate whether the 5'HS3 sequences in these lines are in an open 
chromatin conformation, we performed DNase I hypersensitive site mapping 
was performed on nuclei prepared from 13.5 day transgenic fetal livers 
obtained from three of the single-copy transgenic lines (data not shown), 
including the lowest expressing line C8 (FIG. 7A). FIG. 7B is a map of 
these hypersensitive sites. In FIG. 7A, 8-12 frozen transgenic fetal 
livers were pooled for each line and treated as described (Forrester et 
al., Genes Dev. 4:1637-1649, 1990). In brief, nuclei were prepared by 20 
strokes of a B type Dounce pestle, and 100 .mu.l resuspended nuclei 
aliquots each were digested with increasing volumes (0.5-12 .mu.l) of 80 
.mu.g/ml DNase I (Sigma) for exactly 3 mins at 37.degree.. Reactions were 
stopped, digested with Protease K, phenol/chloroform extracted, and 
ethanol precipitated. The DNA pellet was resuspended in 100 .mu.l water 
after a 45 sec centrifugation, and 20 .mu.l was digested with EcoR1 prior 
to Southern blot analysis and hybridization to the .beta.ivs2 probe as 
described (Kollias et al., 1986, supra). Autoradiography was for 4 days. 
As expected for an expressing transgene, hypersensitive sites were observed 
at 2.7 kbp and 1.5 kbp 5' of the EcoR1 site in the .beta.-globin transgene 
corresponding to the 5'HS3 core and the proximal .beta.-globin promoter 
sequences, respectively. The C8 transgene is slightly more resistant to 
DNase I digestion than the other transgenes (FIG. 7A), suggesting that it 
is located in a particularly inaccessible chromatin region. Nevertheless, 
the C8 transgene chromatin contains the appropriate DNase I hypersensitive 
sites and the promoter is expressed. These data are consistent with the 
presence of a dominant chromatin opening activity residing in the 1.9 kbp 
5'HS3 fragment that not only forms hypersensitive sites on the same 
sequences in the integrated transgene construct as those detected in the 
native chromatin context, but also reproducibly directs transgene 
expression. 
Single-copy transgene expression is predictably and reproducibly directed 
by the 5'HS3 fragment. Although such expression varies among the 
constructs by as much as 6-fold per copy, the activation of transcription 
of the associated transgene is insensitive to position effects and hence 
is position independent. All the 5'HS3 lines express significant 
.beta.-globin levels, and five of them express within a 2-fold range 
(between 20-38% per copy) Moreover, if one compares transgene expression 
for a transgene associated with a chromatin opening domain versus a 
transgene associated with an enhancer only, the lowest expressing 5'HS3 
line produces 60-fold more .beta.-globin mRNA than the non-expressing 
5'HS2 line. Therefore, chromatin domain opening activity enables 
transcription of the associated transgene independent of the integration 
site of the domain/transgene construct in the host cell genome, but does 
not assume identical levels of transgene expression at each integration 
site. 
B) Identification of a CD2 Chromatin Opening Domain 
Studies of the function of the human CD2 gene 3' flanking region has 
revealed the presence of an LCR element which contains regions of tissue 
specific DNase I hypersensitivity. Deletional analysis of this region has 
shown that the LCR function is contained within the 2 kb of 3' flanking 
sequence immediately downstream of the human CD2 polyadenylation signal 
(Lang et al., 1991, Nucleic Acids Research 19:5851). These studies 
demonstrated a correlation between transgene copy number and level of 
specific mRNA within the thymus. Parallel studies using transient 
transfection assays have identified a 900 bp classical enhancer within 
this 2 kb region (Lake et al., 1990, Eur. Mol. Biol. Jour. 9:3129). 
Determination of the location of the chromatin opening domain within this 
2 kb region of the CD2 locus is performed as follows. 
Stepwise deletion of DNA of this 2 kb 3' flanking region is performed and 
the deleted constructs tested for loss of position independent transgene 
expression. Those constructs which retain position independent transgene 
expression, but which have lost full LCR activity and therefore do not 
express the transgene to the full expression level of the complete CD2 LCR 
(i.e., the 2 kb 3' flanking sequence) will contain the CD2 chromatin 
opening domain. The deletion constructs, and testing of the constructs for 
chromatin opening domain activity is described in detail below. 
In order to further define the hCD2 LCR DNA sequences necessary for 
position independent expression, a series of 3' deletion constructs were 
used to generate transgenic mouse lines which are analysed at the DNA 
(Southern blot analysis), RNA (Northern blot analysis) and protein level 
(flow cytometry). The latter analysis allows an estimation of the quantity 
of protein expressed on the cell surface of individual thymocytes and T 
cells as measured by fluorescence intensity using a hCD2 monoclonal 
antibody. T cells are identified by concomitant staining for either Thy-1 
or mouse CD4 and CD8. Transgene copy number is estimated by DNA 
hybridisation as previously described (Greaves et al., 1989 Cell 56: 979); 
quantitation of signal is done using a phosphorimager. A minimum of two 
sets of transgenic lines are compared, each carrying a single copy of each 
deletion construct. A positive control construct, in which position 
independent gene expression is retained, is the CD2 minigene linked to 
only 2 kb of immediate 3' flanking DNA. Position independent expression 
will be indicated at the DNA level and at the protein level in mature T 
cells. Similar analysis of thymocytes and T cells from lymph nodes will 
confirm this result (data not shown). In addition, hCD2 expression may be 
found on the thymocyte and T cell subsets defined by CD4 and CD8 
expression (i.e. CD4-CD8-, CD4+CD8+, CD4+CD8- and CD8+CD4-). 
Most LCRS defined to date have been shown to contain regions of tissue 
specific DNAse I hypersensitivity. Long range mapping has identified two 
regions of DNAse I hypersensitivity downstream of the hCD2 transgene in 
thymocytes (Greaves et al., 1989 Cell 56: 979). For localization of DNase 
I hypersensitive sites, DNA is extracted from transgenic thymocyte nuclei 
which had been treated with increasing concentrations of DNAse I. This DNA 
is then subjected to Southern blot analysis following digestion with 
HindIII which liberates the 2Kb of 3' DNA immediately downstream of the 
hCD2 polyadenylation signal. The blot is hybridised with a probe derived 
from the 5' end of the 2 kb region under investigation. A single large 
band will represent the parent 2 kb restriction fragment; smaller bands 
which appear only after incubation with increasing concentrations of DNAse 
I represent partial digestions of this fragment by DNAse I. Genomic 
molecular weight markers are used to estimate the size of these bands. 
These markers are obtained by digesting untreated hCD2 transgenic DNA with 
restriction enzymes thereby yielding fragments with a known range of 
predictable sizes which hybridise with the probes used. 
The three DNase I hypersensitive sites in the 3' CD2 flanking sequence were 
localized in this way. The position of the HSS was verified by hybridising 
the same blot with a probe from the 3' end of the 2 Kb Hind III fragment. 
The upstream HSS cluster (HSS region 1) coincides with the region known to 
function as a classical enhancer (Lake et al., 1990, supra). 
The function of the downstream HSS cluster (HSS region 3) is investigated 
by generating transgenic mouse lines in which these site is deleted, and 
by generating transgenic mouse lines in which this site is present. 
1. Construction of transgenes 
The generation of the 3' deletion constructs was as previously described 
(Lang et al., 1990, Nucleic Acids Research 19: 5851-5856), except in the 
case of the CD2 1.3 Kb transgene. The immediate 3' flanking 2 kb of hCD2 
DNA was obtained by digesting a plasmid containing the hCD2 minigene 
linked to this 2 Kb fragment with BamHI (cuts distal to the 
polyadenylation signal) and HindIII (cuts 2 Kb 3' to the polyadenylation 
signal). The DNA obtained was gel purified and truncated further by 
digesting to completion with SacI (situated 1.5 Kb 3' to the 
polyadenylation signal). This 1.5 Kb fragment was purified and then 
partially digested with Afl II which yielded a 1.3 Kb fragment (which 
extended from the polyadenylation signal of the hCD2 gene to 1.3 Kb 
downstream). This fragment was purified after gel electrophoresis and 
ligated to a linearised and blunted plasmid (bluescript) containing the 
hCD2 minigene with 4.5 Kb of 5' flanking DNA and no 3' flanking DNA. The 
latter plasmid had been linearised with Bam H1--which lies immediately 3' 
to the polyadenylation signal. E. coli clones were selected after 
transformation and culture and the DNA obtained was screened (using 
asymmetric restriction enzyme digests) to ascertain the orientation of the 
1.3 Kb fragment with respect to the hCD2 minigene. Both orientations (CD2 
1.3 Kb and 1.3 Kb) were lifted out of bluescript with a Sal 1--Not 1 
digest. The fragments were prepared for microinjection as described 
previously (Greaves et al., 1989, Cell 56: 979). 
The generation and screening of transgenic mice is as described previously 
(Greaves et al., 1989, Cell 56: 979) using CDA/Ca, C57/Bl10 and 
CBAxC57/Bll0F.sub.x mice. 
2. Flow cytometric analysis 
For the evaluation of the pattern and level of hCD2 expression, 10.sup.6 
thymocytes, mesenteric lymph mode cells or peripheral blood cells are 
incubated for 30 minutes at 4.degree. C. with CD4 red (Boehringer 
Mannheim), CD8 PE (Catlag laboratories) and FITC conjugated anti-hCD2 
(OKT11) antibodies. Lysis of red cells is done using Becton Dickinson 
lysis solution according to the manufacturers instructions. Cells are 
analysed using a Beckton Dickinson FACS sorter. Three color analysis is 
done using the Lysis II programme with a Hewlett Packard computer. 
3. Preparation of Human CD2 positive and negative thymocytes from 
transgenic mice 
Thymocytes from 3 week old transgenic mice are obtained by teasing the 
thymuses in PBA (PBS with 1% BSA, Sigma). The human CD2 positive cells 
obtained are extracted, after 45 minute incubation with purified OKT11 
mouse anti-human CD2 monoclonal antibody (a gift from Dr. Cantrell ICRF), 
using Dynal magnetic beads coated with Rat anti-mouse IgG1 using the 
procedure described by the manufacturer. 
4. DNase I hypersensitivity mapping 
Nuclei were extracted from both hCD2 positive, hCD2 negative or unsorted 
thymocytes and subjected to DNAse I digestion (Sigma) using the procedure 
described previously (Greaves et al., 1989, Cell 56: 979). The DNA 
extracted was subjected to Southern analysis (Southern, 1975, Journal of 
Molecular Biology 98: 503-517) following digestion with Hind III (for 
mapping within the full hCD2 gene) or BhlII (for mapping in the CD2 1.3 Kb 
transgenic lines). The nitrocellulose blots obtained were hybridised with 
either a) a .sup.32 p labelled hCD2 3' flanking probe extending from the 
polyadenylation signal 500 bp downstream or b) a 3'(NcoI-BamHI) or 5' 
fragment of the hCD2 cDNA. The blots were stripped and rehybridised with a 
700 bp 3' endogenous Thy-1 probe--an Apa I fragment from the 4th 
exon--previously used to map hypersensitive sites within this locus 
[Spanopoulou Phd thesis]. They were analysed using autoradiography and a 
phosphorimager. All cellular manipulations were performed on ice apart 
from the DNAse I digestion which was done at 37.degree. C. 
The 1.3 and 1.5 kb portions of the 2 kb CD2 3' flanking region and the 
corresponding 0.7 and 0.5 kb portions, respectively, are tested in 
association with a transgene, as described above, in single copy 
transgenic mice. Where position independent transgene expression is 
retained in a shortened fragment, a chromatin opening domain is indicated 
if the level of transgene expression is less than about 60% of the level 
of transgene expression in association with the 2 kb CD2 LCR. 
C) Identification of a Class II MHC Chromatin Opening Domain 
A chromatin opening domain of the class II major histocompatibility complex 
(MHC) locus may be identified as follows. The MHC class II LCR is 
described in Carson & Wiles, 1993, Nucleic Acids Research 21:2065-2072. 
The chromatin opening domain of this LCR may be identified as described 
above for the .beta.-globin and CD2 chromatin opening domains. Based on 
knowledge available in the art with respect to this LCR, the chromatin 
opening domain from the class II MHC will be smaller than the fully 
functional LCR described in Carson & Wiles, and larger than a fragment 
containing a deletion of three of the five DNase hypersensitive sites 
mapped in the MHCII LCR, as disclosed in Carson & Wiles, which deletion 
destroys LCR function. 
The class II MHC chromatin opening domain will retain the 
tissue-specificity of the full-length class II MHC LCR, and thus will 
direct transgene expression primarily in B or pre-B cells, and will also 
retain position-independent transgene expression of the full length LCR, 
but will not allow for expression of the transgene to the level conferred 
by the full length LCR. The reduced level of transgene expression 
conferred by the class II MHC chromatin opening domain will be less than 
60% of the level of expression of the transgene when associated with the 
full length corresponding LCR, will likely be less than 40%, and may be on 
the order of 10-25%. 
D) Identification of a Macrophage-Specific Lysozyme Chromatin Opening 
Domain 
A chromatin opening domain of the macrophage-specific lysozyme locus may be 
identified as follows. The chromatin opening domain of the 
macrophage-specific lysozyme LCR or the chromatin opening domain of the 
human lysozyme locus control region are described in Bonifer et al., 1990, 
Euro. Mol. Biol. Org. Jour. 9;2843; and Bonifer et al., 1994, Nucleic 
Acids Research 22:4202-4210. 
A construct useful for testing for the presence of a macrophage-specific 
lysozyme chromatin opening domain may also contain the lysozyme gene 
promoter and a reporter gene. The chicken lysozyme LCR and promoter is 
carried on an 11.8 kb XhoI-SacI fragment from pIII.lyx construct as 
described in Bonifer et al., 1990 supra. This 11.8 kb fragment may be 
shortened to determine the location of its chromatin opening domain, as 
described herein, and the shortened fragment ligated to the reporter gene. 
These chromatin opening domains may be identified according to the 
procedures used, as described herein, for localization of the 
.beta.-globin chromatin opening domain. That is, the macrophage-specific 
lysozyme chromatin opening domain will retain the tissue-specificity of 
the full-length lysozyme LCR, and thus will direct transgene expression 
primarily in macrophages, and will also retain position-independent 
transgene expression of the full length LCR, but will not allow for 
expression of the transgene to the level conferred by the full length LCR. 
The reduced level of transgene expression conferred by the 
macrophage-specific lysozyme chromatin opening domain will be less than 
60% of the level of expression of the transgene when associated with the 
full length corresponding LCR, will likely be less than 40%, and may be on 
the order of 10-25%. 
EXAMPLE IV 
How to Recognize an LCR Subregion That Does Not Contain a Chromatin Opening 
Domain 
An LCR subregion that does not comprise a chromatin opening domain and 
therefore does not fall within the claimed invention may be identified as 
follows. Three subregions of the .beta.-globin locus, i.e., the so-called 
5'HS2 and 5'HS4 regions encompassing .beta.-globin enhancer elements (see 
FIG. 3A-B), as well as a portion of the 5'HS3 region known as footprints 
1-3 (FP1-3, see FIG. 3C), were tested for chromatin domain opening 
activity and determined to lack this activity. 
A small 5'HS2 core element has been shown to be a partial LCR that directs 
copy-number dependent expression of multicopy 5'HS2/.beta.-globin 
transgene concatamers, but fails to direct expression of single-copy 
transgenes (Ellis et al., 1993, supra; Ellis et al., 1993, supra, both of 
which are hereby incorporated by reference). 
The 5'HS2 construct, i.e., the "B" construct (FIG. 8), contains the 
wild-type 1.5 kbp Kpn1-Bg1I 5'HS2 fragment and thus includes the complete 
wild-type 5'HS2 fragment including the core and auxiliary factor binding 
sites in a fragment of about 1.5 kbp. The hypersensitive site was cloned 
into the polylinker 5' of the 800 bp human .beta.-globin promoter or into 
the EcoRV site 3' of the human .beta.-globin gene (exons shown in thick 
black boxes) in GSE 1758. 
Founder adult transgenic mice were generated with the B constructs and 
identified by Southern blotting on tail DNA (FIG. 8). Because the F.sub.0 
generation copy number is difficult to establish reliably due to different 
cells containing different integration events and copy numbers, all 6 
founder lines were bred to nontransgenic animals to obtain nonmosaic 
F.sub.1 fetuses representing 6 different integration events (FIG. 8). The 
copy number of these lines was unambiguously determined by Southern blot 
analysis on fetal head DNA digested with EcoRl and hybridized with probes 
specific for human .beta.-globin (FIG. 9A) or mouse Thy-1 as a loading 
control (FIG. 9B). EcoR1 cleaves the transgene downstream of the probe and 
therefore single-copy transgenes will be visualized as an end fragment of 
random size, and higher copy numbers will usually contain one end fragment 
and a multicopy head-to-tail transgene concatamer. The copy number was 
verified for all lines by Southern blot analysis of Bam H1 digested DNA, 
and transgene intactness was confirmed by PCR analyses of the 5' and 3' 
ends (primer locations shown in FIG. 8; data not shown). Transgene 
intactness was further confirmed by additional Southern blots of BSpH1, 
and BamH1-Xba1 digested DNA for the B lines. By this process, we 
identified a range of copy numbers of intact transgenes including two 
single-copy lines for the B constructs. 
S1 analysis was performed on fetal liver RNA to examine the expression 
status of the transgenes relative to the endogenous mouse .beta.major 
genes. As a positive control, we used RNA from line 72 which contains a 
single copy of the entire human .beta.-globin locus including the 20 kbp 
LCR fragment, and expresses human .beta.-globin mRNA at about 50% the 
level produced by the two mouse .beta.major genes or 100% per copy. In the 
S1 analysis shown in FIG. 10, human .beta.-globin expression by line 72 
was calculated to be at least 90% per copy. Globin genes that are not 
linked to LCR sequences express at less than 0.1% per copy. 
The data shown in FIG. 10 indicates that the 5'HS2 construct is not 
sufficient to reproducibly obtain expression from a single-copy 
.beta.-globin transgene. The results show that random integration of the 
construct into inactive or active regions of a chromosome result in 
undetectable and detectable transgene expression, respectively, but that 
detectable expression is not obtained in a predictable and reproducible 
manner. In FIG. 10, S1 analysis of fetal liver RNA from these mice 
detected human .beta.-globin expression at less than 1% per copy in each 
of a single-copy and a two copy line, and 3% per copy in the 5 copy line. 
A level of 8% per copy was detected in one single-copy line and a level of 
0% per copy in the other single copy line. The seven copy lines both 
expressed at about 23% per copy. These data demonstrate that the full 
length wild-type 5'HS2 fragment cannot reproducibly activate single-copy 
transgene expression. That is, if the 5'HS2/transgene construct randomly 
integrates into a region of chromatin that is open, and thus is expressed, 
then transgene expression may be randomly obtained in a single copy 
construct. However, if the same construct integrates into a closed region 
of DNA, then the 5'HS2 region is unable to open the chromatin and allow 
for transgene expression. Thus, reproducible chromatin opening activity is 
not found within the 5'HS2 region. Testing of the 5'HS4 construct (FIG. 
3B) revealed similar results as the 5'HS2 fragment with respect to an 
inability to reproducible activate single copy transgene expression. In 
addition, other experiments demonstrated that footprints 1-3 of 5'HS3 core 
are unable to reproducibly activate single-copy transgene expression (data 
not shown). 
MECHANISM OF ACTION 
Without being bound to any one theory, it is postulated that the 
integration-site independence demonstrated by LCRs is attributable to two 
factors. First, an LCR is able to transform the chromatin surrounding it 
into an open chromatin structure. The chromatin opening activity is 
essential in order to actuate transcription regardless of the site of 
integration, but is in itself not sufficient to give rise to physiological 
levels of transcription as the chromatin opening domains do not 
necessarily have enhancer activity. 
Secondly, an LCR contains powerful enhancer elements, which are not 
chromatin opening domains, which in single copy integrants cannot give 
rise to physiological levels of transcription when the enhancer elements 
are not associated with the LCR subregions of the invention. The level of 
enhancement is so high that any position effects due to the surrounding 
chromatin environment are effectively masked. 
The chromatin opening domains of the invention, therefore, are capable of 
actuating transgene expression independent of the site of integration of 
the transgene into host cell chromatin. However, some chromatin opening 
domains of the invention, i.e., those which have been physically separated 
from an associated enhancer, are not able to confer reproducible 
physiological level expression because they do not possess the full level 
of transcriptional activity of a fully-functional LCR. The result is that, 
while significant expression is always observed, the level of such 
expression can vary more than that observed with an LCR which includes its 
enhancers. 
Actuation of transcription occurs when a transgene is integrated in the 
form of open chromatin in the genome of the host cell. The transgene is 
always in a fundamentally active state and therefore susceptible to 
control by conventional transcription-regulating factors. In contrast, in 
the absence of the chromatin opening domains of the invention, transgenes 
often integrate into regions of chromatin which have a closed 
conformation, which confers a fundamentally inactive state on the 
transgene. There is thus a qualitative difference in the state of the 
transgene depending on the presence of a chromatin activating domain. In 
terms of quantitative expression, even the lowest levels of transcription 
observed when using the domains of the invention (6%) are substantially 
higher than the levels of expression observed without a chromatin opening 
domain (typically less than 0.1%). 
The observed variation is a position effect and depends on the chromatin 
environment in which the transgene integrates. Thus, if the transgene 
integrates into a highly active chromatin region in the vicinity of a 
powerful enhancer, a physiological level of expression will be observed. 
Conversely, if the transgene integrates into a very inactive area of 
chromatin, as occurs in the majority of cases, the absence of enhancer 
function will mean that the level of expression observed will be lower 
than a physiological level. 
In both cases, however, the presence of the chromatin opening domain will 
ensure that the transgene is expressible at physiological levels given the 
provision of appropriate enhancer functions. 
The complete .beta.-globin LCR is composed of multiple separable elements 
surrounding the four HS sites, 5'HS3 being responsible for the dominant 
chromatin opening activity of the LCR, and therefore being the primary 
regulator of transcription activation in vivo. 5'HS3 alone confers on 
average 26% expression per copy on single copy transgenes, i.e., a 
non-physiological level of expression, indicating that in the complete LCR 
additional transcriptional enhancer activity is provided by the other HS 
sites, including the 5'HS2 enhancer. Because chromatin domain opening 
activity is separable from enhancer activity, but enhancer activity is not 
evident without chromatin opening activity, enhancer activity can be said 
to be secondary or auxiliary to the essential chromatin opening activity 
of the 5'HS3 element. 
The approach described herein for evaluating LCR sequences in single-copy 
transgenic mice provides a system for evaluating DNA elements that 
regulate mammalian gene expression. That is, in single-copy transgenic 
mice, enhancer and other LCR elements are functionally distinguishable. 
Moreover, expression from constructs designed for retrovirus or 
adeno-associated virus vectors can also be evaluated most reliably in 
single-copy transgenic mice. By including a dominant chromatin opening 
domain, such as that residing in 5'HS3, in a gene construct, it should be 
possible to express every single-copy vector integration event in a 
desired tissue. 
EXAMPLE V 
How to Identify and Characterize Other Chromatin Opening Domains of the 
Invention 
An LCR subregion comprising a chromatin opening domain may be identified 
and isolated by one of skill in the art using techniques known in the art 
and described herein according to the following method of identification 
and isolation. 
One of skill in the art would be able to determine if a chromatin opening 
domain exists in a region of DNA by performing the following testing 
procedure. First, a region of DNA that is suspected of containing 
chromatin opening domain activity is isolated. Such a candidate region 
would, of course, reside within a locus control region. Locus control 
regions are associated with tissue-specifically expressed genes, and have 
been identified and isolated for several different loci, as discussed 
above. One criterion for the identification of a chromatin opening domain 
within a locus control region is its association with a DNase I 
hypersensitive site. Minimally, a chromatin opening domain will be 
associated with one such site; however, the domain may be associated with 
several such sites, for example, two, three or four hypersensitive sites, 
depending upon the length of DNA which the domain occupies and the number 
of hypersensitive sites clustered near or within the domain. A candidate 
chromatin opening domain may be provided, for example, as a fragment of 
DNA (e.g., a restriction fragment) from a known and isolated locus control 
region. Therefore, several candidate domains may be provided and tested 
simultaneously simply by digesting an LCR into two or more fragments, each 
fragment being an LCR subregion. 
Second, once a candidate chromatin opening domain is provided as a DNA 
fragment, the fragment may be linked to a reporter gene to generate a 
construct. The reporter gene will be expressible in that it will be 
operably associated with a promoter that allows for gene expression. The 
reporter gene may be chosen by virtue of its encoding an easily assayable 
product, or it may simply be the gene that is naturally associated with 
the LCR from which the candidate chromatin opening domain is derived. 
Third, the construct is then introduced into a host cell, usually, but not 
always, a mammalian host cell. The host cell may be a cell line that is 
carried in culture, and therefore the construct will be introduced via 
known procedures such as transfection, transduction or microinjection into 
a cultured host cell line without selection; e.g., using retroviruses. 
Alternatively, the host cell may be cells of a transgenic animal, in which 
case the construct is introduced into the animal using known procedures 
for making transgenic animals, as disclosed herein. Once the construct is 
introduced into the host cell, it then integrates into the host cell 
genome, and the reporter or test gene is known as a transgene. 
Fourth, the candidate chromatin opening domain is then tested in those 
cells containing a single copy of the transgene for chromatin opening 
domain activity. As described in detail herein, a chromatin opening domain 
reproducibly actuates transcription of a transgene with which it is 
associated. Thus, in order to ascertain reproducible actuation of 
transcription, one of skill in the art would test a number of transgenic 
cell lines or transgenic animals, each cell line or animal representing an 
independent integration event, for expression of the transgene. Actuation 
of transgene transcription is considered reproducible, and thus 
integration-site independent, if the transgene is expressed in three 
different transgenic cell lines or transgenic animals. However, more than 
three cell lines or animals may be tested. 
The actual level of expression of the transgene in the transgenic cell line 
or animal will be determined by measuring the amount of RNA expressed by 
the transgene, as described in detail herein, and comparing that amount to 
the amount of RNA expressed by the endogenous host cell gene. The 
endogenous host cell gene will be defined by virtue of its association 
with an endogenous LCR that is considered equivalent to the LCR from which 
the candidate domain is derived. An equivalent endogenous LCR is not 
difficult to identify and is defined in the Summary of the Invention 
above. The amount of test gene RNA is measured as a percentage of the 
amount of endogenous gene RNA. The percentage is arrived at by forming a 
ratio of the amount of test gene RNA over the amount of endogenous gene 
RNA times 100. Of course, differences in specific activities of the two 
RNAs must be taken into account. Therefore, for instance, if the specific 
activity of the test gene RNA is twice that of the endogenous gene RNA, 
then the amount of endogenous gene RNA is multiplied by two prior to 
calculating the percentage: (test gene RNA/2.times.endogenous gene 
RNA).times.100. This simple formula (I) provides the total expression 
levels of test gene and endogenous gene in a host cell. 
The results may also be expressed according to a slightly different 
formula. That is, since the test gene may be introduced in single copy or 
multiple copies in the host cell genome, and the endogenous gene is 
normally present in two copies in the host cell genome, the results may 
also be expressed as expression of single copy of endogenous gene per 
single copy of test gene. This is calculated as two endogenous genes 
divided by the number of transgenes, multiplied by the percent expression: 
(2.times.endogenous gene/.times.number test genes).times.(% expression=I). 
The candidate chromatin opening domain is determined to possess chromatin 
opening domain activity according to two criteria. First, the RNA 
comparison must provide results which show that the candidate domain 
reproducibly provides at least a minimum level of test gene expression 
(i.e., minimally, equal to or above a 1% level), but does not provide the 
full level of expression conferred by the complete LCR (i.e., less than 
full level expression corresponding to less than about a 90% level). This 
level of test gene expression will be reproducible because it occurs 
independent of the integration site of the test gene in the host genome. 
Therefore, a candidate chromatin opening domain will be considered to fall 
within the first criterion defining a chromatin opening domain if the 
expression of the test gene is reproducibly between 1% and 80% of the 
level of expression of the endogenous gene. 
The second criterion for defining a chromatin opening domain relates to the 
ability of the domain to "open" the chromatin in the region of the test 
gene. Such opening activity is determined by DNase I hypersensitivity. 
That is, if the candidate domain possesses a DNase I hypersensitive site 
both in its native context and in its non-native context integrated in the 
host cell genome, then it falls within the second criterion defining a 
chromatin opening domain. 
DNase I hypersensitivity is defined above in the Summary of the Invention. 
A DNA fragment is considered to possess a DNase I "hypersensitive site" if 
the fragment contains a site that is preferentially cut in a DNase I 
sensitivity assay. Preferential cutting is usually ascertained by 
detecting a DNase I-cut band in a Southern blot. A DNA fragment lacks a 
DNase I hypersensitive site where preferential cutting does not occur; 
i.e., instead of detecting a discrete band after digestion with DNase I, a 
smear of lower molecular weight DNA may be detected representing random 
cutting events at longer incubation times or higher concentrations of 
DNase I. Although the parameters for performing DNase I sensitivity assays 
may vary, e.g., the DNase I concentration, specific activity, and 
temperature and time of digestion, the identification of hypersensitive 
sites (as distinguishable from sensitive sites) is well-known and 
well-practiced in the prior art, and thus will be routine identification 
using variations on the above parameters. 
In order to fall within the definition of chromatin opening domain, as 
defined herein, a candidate domain must fulfill both the first and second 
criteria described above, that is, the domain must reproducibly confer 
expression on the test gene when the domain/test gene construct is carried 
in single copy in the host cell genome, the test gene expression being 
about 1-80% of endogenous gene expression, and the domain must be 
associated with one or several DNase I hypersensitive site(s). 
EXAMPLE VI 
How to Use Constructs of the Invention 
Use of an LCR subregion containing a chromatin opening domain, as described 
herein, is particularly desirable for the following reasons. First, 
because the LCR subregion/transgene construct is smaller than the 
full-length LCR, methods of transfer of the construct are useful that were 
not previously possible using full-length LCR constructs. These methods 
include the use of viral vectors, which allow for transfer to an animal of 
an LCR subregion/transgene construct but not for a much larger full-length 
LCR/transgene construct. Thus, production of a transgenic animal solely at 
the animal embryo stage by microinjection of the construct is not required 
by the invention. This represents a considerable advantage where a large 
transgenic animal, e.g., a cow or bull, is produced, as it is difficult to 
produce such animals via embryo microinjection techniques but not via 
viral infection. 
Second, the use of LCR subregion/transgene constructs allows for 
tissue-specific production of a desired protein by virtue of the 
tissue-specific nature of transgene expression conferred by the domain. 
Third, the use of constructs of the invention allow for production of a 
controlled non-physiological level of the trans-protein by virtue of the 
integration-site independence of transgene expression conferred by the 
domain and the ability of the transgene to express when integrated in 
single copy. 
The invention thus provides domains for use in medicine; that is, for use 
in the manufacture of human proteins from animals. 
An LCR subregion comprising a chromatin opening domain may be used for 
production of therapeutically useful proteins in a number of ways, as 
follows. 
For example, an LCR subregion of the invention may be used in transgenic 
animals where integration-site independent expression of a transgene is 
desired. In addition, it may be desirable to produce a protein in a 
specific tissue, e.g., blood tissue or mammary tissue, using a single copy 
of the protein-encoding gene. 
For example, where it is desirable to produce human .beta.-globin in blood 
tissue of a transgenic animal, a chromatin opening domain of the 
invention, e.g., the HS3 domain, linked to the human .beta.-globin gene, 
may be transferred to the animal either using conventional transfer of 
genetic material at the early embryo stage or using viral transfer 
vectors. A level of human .beta.-globin that is less than the 
physiological level of the animal's .beta.-globin may be necessary, for 
example, to obtain proper processing or glycosylation of the foreign 
protein. A non-physiological level of human .beta.-globin may be obtained 
by transferring the HS3/human .beta.-globin construct described herein to 
the animal and purifying human .beta.-globin from the animal's blood 
tissue. 
Similarly, where it is desirable to produce human growth hormone in a 
transgenic animal, the level of growth hormone produced in the animal must 
be carefully controlled due to the deleterious effects of over-production 
of the hormone. Such control may be achieved by transferring a chromatin 
opening domain of the invention coupled to a gene encoding human growth 
hormone to an animal genome without concern for whether the construct will 
integrate into an inactive position in the genome. The inventive domain 
will open the chromatin at the site of integration of the construct in the 
host cell genome to allow for expression of the human growth hormone gene 
at a controlled level. Human growth hormone may then be purified from the 
transgenic animal. 
In addition, for example, where it is desirable to produce a human blood 
clotting factor, e.g., human factor VIII, IX or von Willebrand factor, in 
the mammary tissue of an animal, or other proteins, e.g., albumin, 
lactoferrin, a transgenic animal may be produced using a chromatin opening 
domain coupled to the gene encoding the factor. The factor may then be 
purified from milk produced by the animal. 
Methods for producing transgenic animals via germ line or viral transfer 
events, e.g., mice, pigs, chickens, sheep, cows, bulls, etc., are 
well-known and documented in the art. 10 A domain of the invention also is 
useful in gene therapy-related applications, for example, when a gene 
construct comprising a domain of the invention is integrated in single 
copy into the genome of a host cell. 
At present, in order to definitively test expression levels from a 
potential gene therapy construct, the construct must be inserted into an 
integration competent viral vector, transferred into a helper cell line to 
assemble stable high-titer virus, and then successfully transduced into 
mouse hematopoietic stem cells (for review see Miller, Nature 357:455-460, 
1992). Long-term repopulation of mice by the infected stem cells finally 
produces mature differentiated cells that can be assessed for expression 
of the transduced gene. 
An alternative method is to test expression from a potential gene therapy 
construct present in transgenic mice at a single copy before the 
difficulties of assembling a high-titer virus are confronted. In the case 
of .beta.-globin vectors, expression is evaluated in erythroid cells 
derived from stem cells that contain the construct as a single-copy 
transgene, and only those .beta.-globin constructs that express 
appropriately and reproducibly need be packaged into virus for use in gene 
therapy. 
Since the discovery of LCRs, LCRs have been indicated for use in gene 
therapy. LCR subregions of the invention are also useful for gene therapy, 
for example, treatment of cardiovascular diseases, AIDS, inherited or 
acquired genetically based diseases, hemaglobinopathies, cystic fibrosis, 
severe combined immune deficiency diseases, lysosomal storage disease, 
muscle diseases, etc. In the case of the .beta.-globin 5'HS3 region, in 
particular, gene therapy treatments of diseases associated with blood 
tissue disorders are useful. 
One difficult aspect of gene therapy is reproducibly obtaining high-level, 
tissue-specific, and long-term expression from genes transferred into stem 
cells (for reviews see Mulligan, Science 260:926-932, 1993; Dillon, Trends 
Biotech. 11:167-173, 1993). LCRs confer such position-independent 
activation on transgenes. Additional important considerations are that the 
gene system used should be as compact as possible, in view of the need to 
package the gene into available delivery systems as well as the need to 
minimize possible adverse effects on the genome of the recipient cell, and 
furthermore that the gene should be active in single copy in the host cell 
genome. This latter consideration is important because many viral systems 
currently proposed for the delivery of therapeutic genes integrate in 
single copy in host cells. Thus, LCR subregion/transgene constructs of the 
invention allow for carefully controlled expression of the transgene 
because they allow for single-copy gene expression. 
The following examples provide specific uses of a construct according to 
the invention. 
EXAMPLE VII 
Targeted Delivery of Chromatin Opening Domain Construct to Treat Hemoglobin 
Disorders, Sickle-Cell Anemia and Thalassemia 
Blood disorders such as severe hemoglobin (Hb) disorders, sickle cell 
anemia and thalassemia are treatable according to the invention using a 
retroviral vector comprising the chromatin opening domain from the 
.beta.-globin locus control region and a human .gamma.-globin gene. Miller 
et al. (1994 supra) describes infection of CD34+ human hematopoietic cells 
with a recombinant rAAV construct containing the human .gamma.-globin gene 
the HS2, HS3, and HS4 sites of the human .beta.-globin locus, and 
subsequent expression of the .gamma.-globin gene. 
The present invention contemplates use of the HS3 site from the 
.beta.-globin locus in a gene therapy construct to obtain position 
independent expression of a functional gene to treat a blood disorder. 
Chromatin opening domain-containing constructs of the invention can thus 
be used to transfer a globin gene into repopulating stem cells with 
subsequent expression in erythroblasts in vivo. 
A human .beta.-globin COD construct useful for treatment of a blood 
disorder may be prepared as follows. The human .beta.-globin COD (fragment 
HS3), and the .sup.A .gamma.* globin gene (Sorrentino, B. P. (1990) Ann. 
N.Y. Acad. Sci. 612, 141-151) are subcloned into pUC007 (Ney, P. A. (1990) 
Genes Dev. 4, 993-1006). A Bgl II/Sal I fragment of this construct is 
subcloned into pUC008 (Walsh, C. E. (1992) Proc. Natl. Acad. Sci. USA 89, 
7257-7261), which is then digested with Nhe I and ligated to the Xba I 
fragment of pSUB201. The pSUB201-derived AAV inverted terminal repeats 
will flank the human .beta.-globin COD. The plasmid construct is 
cotransfected with the complementing plasmid, pAAV/ad (Samulski, R. J. 
(1989) J.Virol. 63, 3822-3828) into 293 cells previously infected with 
adenovirus type 5 to make the recombinant AAV. Preparation of cell lysates 
containing rAAV, Hirt extracts, and Southern blot analyses are described 
in Walsh, C. E. (1992) Proc. Natl. Acad. Sci. USA 89, 7257-7261; Samulski, 
R. J. (1989) J. Virol. 63, 3822-3828; Hirt, B. (1967) J. Mol Biol. 26, 
365-369. Any portion of the globin gene may be used as a probe. All rAAV 
cells lysates are concentrated by ultrafiltration using a model 8400 stir 
cell apparatus and XM300 membrane (Amicon) prior to heat inactivation of 
adenovirus (56.degree. C., 30 min). The final volume of concentrated cell 
lysate will be .apprxeq.1 ml per 10-cm.sup.2 dish of 293 cells used for 
cotransinfection. 
rAAV particle titer is estimated as follows. A variation of previous assays 
(Samulski, R. J. (1989) J. Virol. 63, 3822-3828) may be used to estimate 
particle number. Twenty microliters of rAAV cell lysate is incubated 
(37.degree. C., 1 hr) with 200 units of DNase (Boehringer Mannheim) in a 
final volume of 200 .mu.l (20 mM Tris-HCl, pH 8.0/10 mM MgCl.sub.2 
buffer). DNase-protected particle (DPP) viral DNA is extracted with RNA 
STAT-60 (Tel-Test, Friendswood, Tex.) using the manufacturer's protocol 
with a final volume of 20 .mu.l. This technique favors recovery of the low 
molecular weight single-stranded DNA genome of the rAAV vector particles. 
The polymerase chain reaction (PCR) will generate a fragment spanning the 
junction between HS3 and the .sup.A .gamma.* globin gene in the 
recombinant globin locus. PCR conditions are as follows: 23 cycles; 
95.degree. C./1 min, 58.degree. C./1 min. 72.degree. C./1.5 min; 5' 
primer, 5'-TCTTCAGCCTAGAGTGATGAC (SEQ ID NO:10); 3' primer, 
5'-ATAGTAGCCTTGTCCTCCTC (SEQ ID NO:10). 
CD34.sup.+ selected progenitor cells are prepared and transduced as 
follows. Human peripheral blood mononuclear cells are obtained by 
hemapheresis of a patient with Hb SS disease. A Ceprate kit (CellPro, 
Bethell, Wash.) is used for CD34.sup.+ cell enrichment according to the 
manufacturer's protocol. One thousand CD34+selected cells are exposed to 
500 .mu.l of rAAV-containing cell lysate (10.sup.6 particles) in a total 
volume of 1000 .mu.l of tissue culture medium (Dulbecco's modified Eagle 
medium, 15% fetal calf serum, 50 ng of interleukin 6 per ml, and 100 ng of 
stem cell factor per ml). After an overnight exposure with gentle rocking 
at 37.degree. C. in 5% CO.sup.2, the cells are resuspended to 10.sup.3 
cells per ml and plated at 1000 cells per plate in methylcellulose 
containing growth factors (10 ng of granulocyte/macrophage 
colony-stimulating factor per ml, and 5 units of erythropoietin per ml). 
Cells are incubated at 37.degree. C. in 5% CO.sub.2 for 13-19 days prior 
to analysis of progenitor derived colonies. 
The COD construct is transferred and expressed as follows. RNA extraction 
from individual colonies is performed by placing each colony (&lt;10 .mu.l of 
methylcellulose) in 250 .mu.l of Stat-60 (Tel-test) according to the 
manufacturer's protocol and maintained at -70.degree. C. until reverse 
transcriptase PCR (RT-PCR) analysis. RT-PCR reagents and the thermal-cycle 
are obtained from Perkin-Elmer. The reverse transcriptase reactions 
(42.degree. C./30 min, 95.degree. C./5 min) are performed as single or 
double volume mixtures, followed by single or matched PCRs (35 cycles; 
95.degree. C./1 min. 60.degree. C./1 min) using the appropriate primers. 
RNA-derived PCR mixtures, which included [.sup.32 P] CTP, are 
electrophoresed on 10% denaturing polyacrylamide gels and dried prior to 
autoradiogram or Phosphorimager analysis. Comparison of the polyacrylamide 
gel band intensities is made using the densitometry function of a 
Phosphorimager (Molecular Dynamics) High-performance liquid chromatography 
HPLC is used for Hb analysis as described (Fibach, E. (1993) Blood 42, 
162-165). 
A construct according to the invention, containing a chromatin opening 
domain from the .beta.-globin locus, an expressible .gamma.-globin gene, 
and a means for delivering this DNA to a target cell, e.g., recombinant 
AAV, is useful for delivering a single copy of the .gamma.-globin gene to 
such cells to restore a globin deficiency. For example, the construct 
described above may be used to treat sickle cell anemia, wherein there 
exists a deficiency of normal globin due to the presence of a mutation in 
the globin gene which consists of a single amino acid substitution. In 
individuals who are homozygous for the sickle hemoglobin abnormality, this 
single amino acid substitution brings about aggregation of hemoglobin 
molecules into polymers when the oxygen tension is lo, thus distorting the 
normally pliable discoid red cells into a charcteristic sickled shape. A 
construct of the invention comprising a chromatin opening domain may be 
used to restore normal globin synthesis in that the integration of a 
single copy of the construct would result in synthesis of a level of 
normal globin that is significantly lower (e.g., 25%) than if the complete 
LCR were present, but sufficient in amount to prevent the aggregation of 
defective globin molecules which results in cell sickling. 
Alternatively, a construct of the invention that comprises a chromatin 
opening domain could be used to treat thalassemias, which are 
characterized by deficient synthesis of the .alpha.- or .beta.-globin 
chains. A chromatin opening domain is particularly useful in the context 
where a non-physiological level, i.e., less than the normal amount, of 
synthesis of a protein occurs. For example, a construct containing the 
.beta.-globin COD and the .alpha.- or .beta.-globin gene may be used to 
restore synthesis of the .alpha.- or .beta.-globin chains in that the 
presence of a single copy of the construct should be sufficient to produce 
enough .alpha.- or .beta.-globin to bring the amount of that molecule to 
normal (physiological) levels within the cell. 
EXAMPLE VIII 
Targeted Delivery of Chromatin Opening Domain Construct to Primary T 
Lymphocytes and Primary and Cultured Tumor Cells 
In this Example, a DNA construct containing a chromatin opening domain and 
an expressible gene, according to the invention, can be delivered to 
lymphocytes and tumor cells using an AAV-based plasmid, containing AAV 
terminal repeats, and cationic liposomes as carrier molecules. This 
delivery system is described by Philip et al., Molecular and Cellular 
Biology, 1994, 14;2411. This system takes advantage of the simple carrier 
system of lipofection and the stable inheritance capability of the AAV 
plasmid. Therefore, any construct according to the invention may be 
delivered to a selected group of cells utilizing this complex of DNA and 
liposome. A chromatin opening domain derived from an LCR will be selected 
for its tissue specificity; i.e., a CD2 COD for T-cells, an MHC II COD 
from a class II MHC LCR for immune cells such as pre-B cells; a COD from a 
lysozyme LCR for macrophages. Delivery of a construct to primary T-cells 
and primary tumor cells, described below, is meant to be representative 
and not limiting as to cell type or tissue specificity. 
A construct according to the invention may include a chromatin opening 
domain from the CD2 LCR. An approximate 2.0 kb region of the CD2 locus is 
described in Lang et al. (1991, Nucleic Acids Res. 19;5851). This 2.0 kb 
region has been shown by Lang et al. to be sufficient for position 
independent and copy number dependent expression of the CD2 gene in a 
transgenic context. That is, this 2.0 kb region of the CD2 locus is fully 
functional LCR. The chromatin opening domain of the CD2 LCR may be 
localized by testing regions of the 2 kb fragment for position 
independence, as disclosed herein. Once localized, the CD2 COD may be 
inserted into the plasmid pSSV9/CMV-IL2, described in Philip et al., 1994, 
supra. This plasmid contains the human interleukin-2 (IL-2) gene and the 
immediate-early promoter-enhancer element of the human cytomegalovirus 
(CMV) flanked by AAV terminal repeats. Where a reporter gene is desired or 
useful, the CD2 COD may be inserted into the plasmid pA1CMVIX-CAT (Philips 
et al., 1994 supra), which contains the CMV immediate-early promoter 
enhancer sequences and some intervening sequences with splice acceptor 
sequences derived from an immunoglobulin G variable region (pOG44; 
Strategene, La Jolla, Calif.), the bacterial chloramphenicol 
acetyltransferase (CAT) gene, and the simian virus 40 late polyadenylation 
signal flanked by AAV terminal repeats in a pBR322 vector. Plasmid DNA is 
isolated by alkaline lysis and ammonium acetate precipitation followed by 
treatment with DNase-free RNase, phenol-chloroformisoamyl extractions, and 
ammonium acetate precipitation (Ausubel et al., Current Protocols in 
Molecular Biology, John Wiley & Sons, Inc., New York, 1993). 
The rat prostate cell line R3327 and bladder cell line MBT-2 (ATCC) are 
maintained in RPMI 1640 medium supplemented with 5% fetal bovine serum 
(FBS). Cell line 293 is a human embryonic kidney cell line that is 
transformed by adenovirus type 5 (Graham et al., J. Gen. Virol. 36:59-72, 
1977). This cell line is grown in Dulbecco modified Eagle medium 
supplemented with 10% FBS. 
Primary lung, ovarian, and breast tumor cells are obtained from the solid 
tumors of patients. The tumor samples are minced into small pieces and 
digested in 200 ml of AIM V medium (GIBCO, Grand Island, N.Y.) 
supplemented with 450 U of collagenase IV (Sigma, St. Louis, Mo.) per ml. 
10.8 Klett units of DNase I (Sigma) per ml, and 2,000 U of hyaluronidase V 
(Sigma) per ml (Topolian et al., J. Immunol. 102:127-141, 1987). After 1 
to 2 h of digestion, cells are homogenized with a glass homogenizer 
(Bellco, Vineland, N.J.). Cells are washed three times in DPBS-CMF 
(Whittaker, Walkersville, Md.) Lymphocytes are separated from nonlymphoid 
cells by capture on a MicroCELLector-CD5/8 device (Applied Immune Sciences 
(AIS), Santa Clara, Calif.). The microCELLectors are polystyrene devices 
containing covalently immobilized monoclonal antibodies for selection of T 
cells. Nonadherent cells (mainly tumor cells) are removed and cultured in 
RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 U of 
penicillin-streptomycin per ml. and 10% FBS. Tumor cells are cultured for 
2 to 4 weeks prior to transfection. 
Peripheral blood mononuclear cells from healthy controls are isolated from 
buffy coats (Stanford University Blood Bank, Stanford, Calif.) by using 
Lymphoprep (Robbins Scientific, Sunnyvale, Calif.). T cells or T-cell 
subsets are further isolated with AIS MicroCellectors. Briefly, peripheral 
blood mononuclear cells are resuspended at 15.times.10.sup.6 cells per ml 
in 0.5% Gamimmune (Miles, Inc., Elkhart, Inc.) and loaded onto the washed 
CD3, CD4, or CD8 AIS MicroCELLectors. After 1 h. nonadherent cells are 
removed. Complete medium (RPMI 1640 medium [Whittaker] containing 10% FBS, 
2 mM L-glutamine, and 100 U of penicillin-streptomycin per ml) is added to 
the adherent cells in the MicroCELLectors. After 2 to 3 days in a 5% 
CO.sub.2, 37.degree. C. humidified environment, adherent cells are removed 
and prepared for transfection. 
Small unilamellar liposomes are prepared from the cationic lipid 
dimethyldioctadecylammonium bromide (DDAB) (Sigma) in combination with the 
neutral lipid dioleoylphosphatidylethanolamine (DOPE) or cholesterol 
(Avanti Polar Lipids, Alabaster, Ala.). Lipids are dissolved in 
chloroform. DDAB is mixed with DOPE or cholesterol in either a 1:1 or 1:2 
molar ratio in a round-bottomed flask, and the lipid mixture was dried on 
a rotary evaporator. The lipid film is rehydrated by adding sterile 
double-distilled water to yield a final concentration of 1 mM DDAB. This 
solution is sonicated in a bath sonicator (Laboratory Supplies, 
Hicksville, N.Y.) until clear. Liposomes are stored at 4.degree. C. under 
argon. 
For the preparation of recombinant AAV (rAAV) stocks, 293 cells are split 
and grown to approximately 30 to 50% confluence. At this time, the cells 
are infected with adenovirus type 5 and incubated at 37.degree. C. After 2 
to 4 h, the infected cells are cotransfected with 10 .mu.g of plasmid and 
10 of the rep capsid complementation plasmid, p.DELTA.Bal, per 
100-mm-diameter tissue culture dish (0.5.times.10.sup.7 to 
1.times.10.sup.7 cells). Calcium phosphate coprecipitation is used for 
transfection (Hermonat et al., Proc. Natl. Acad. Sci. USA 81:6466-6470, 
1984). At 12 to 18 h after transfection, the medium is removed from the 
cells and replaced with 5 ml of Dulbecco modified Eagle medium containing 
10% FBS. At 48 to 72 h posttransfection, AAV is harvested as follows. The 
cells and medium are collected together and freeze-thawed three times to 
lyse the cells. The medium-cell suspension is then centrifuged to remove 
cellular debris, and the supernatant is incubated at 56.degree. C. for 1 h 
to inactivate adenovirus (Hermonat, supra; Tratschin et al., Mol. Cell. 
Biol. 5:3251-3260, 1985). After heat inactivation, the viral supernatant 
is filtered through cellulose acetate filters (1.2-.mu.m pore size). Viral 
stocks are then stored at -20.degree. C. rAAV stock containing a 10.sup.4 
viral titer is used to infect cells. 
Cellular transfection is performed as follows. For primary tumor cells and 
tumor cell lines, 10.sup.6 cells are plated in 2 ml of serum-free medium 
per well of a six-well dish. Plasmid DNA (10 .mu.g) is mixed with 30 nmol 
of total lipid as liposomes composed of DDAB and DOPE in a 1:2 molar 
ratio. Serum-free medium (0.5 ml) is added to the liposome-DNA complex, 
which is then transferred to the cells. The cells are incubated at room 
temperature for 5 min. and then fetal calf serum is added to the cells to 
yield a final concentration of 5% fetal calf serum. For T cells, 
5.times.10.sup.6 to 10.times.10.sup.6 cells are plated in 1 ml of 
serum-free medium per well of a six-well dish. Plasmid DNA (50 .mu.g) was 
mixed with 100 nmol of total lipid as liposomes composed of DDAB and DOPE 
or cholesterol in a 1:1 molar ratio. The transfections are then performed 
as above. 
IL-2 is assayed as follows. Cells are counted, 10.sup.5 cells are plated in 
1 ml per well of a 24-well plate. The following day, supernatants are 
collected and assessed by using a Quantikine IL-2 enzyme-linked 
immunosorbent assay (ELISA) kit from R & D Systems (Minneapolis, Minn.). 
IL-2 levels are expressed as picograms per mililiter of the supernatant. 
Intracellular IL-2 is assayed as follows. Transfected cells are stained at 
various time points for intracellular IL-2 protein levels by a modified 
flow cytometry procedure (Jung, et al., J. Immunol. Methods 159:197-207, 
1993). Cells are harvested, washed with DPBS-CMF (Whittaker) and 
resuspended at 10.sup.6 cells per ml in cold 1% paraformaldehyde (Sigma) 
in DPBS-CMF for 10 min at 4.degree. C. Cells are washed with DPBS-CMF and 
resuspended in cold DPBS-CMF containing 0.1% saponin 9sigma) and 10% FBS 
(HyClone, Logan, Utah) for 10 min at 4.degree. C. Cells are then washed 
with cold saponin buffer and stained with mouse anti-human IL-2 antibody 
(Genzyme, Cambridge, Mass.) for 15 min at 4.degree. C. Cells are washed 
with cold saponin buffer and stained with a fluorescein 
isothiocyanate-conjugated goat anti-mouse F(ab').sub.2 second-step 
antibody (Caltag, South San Francisco, Calif.) for 15 min at 4.degree. C. 
After washing in saponin buffer, cells are washed with DPBS-CMF and 
resuspended at 10.sup.6 cells per ml for flow cytometric analysis. Flow 
cytometry is performed with a FACScan (Becton Dickinson, Milpitas, 
Calif.). 
To measure CAT activity, the transfected cells are collected, washed twice 
with phosphate-buffered saline, and then resuspended in 0.25 M Tris. pH 
7.8. Cell extracts are obtained by three consecutive freeze-thaw cycles 
followed by centrifugation at 16,000.times.g for 5 min. Protein 
concentrations of extracts are measured by a Coomassie blue G250-based 
assay (Bio-Rad. Richmond, Calif.), and protein concentrations were 
normalized. A volume of extract is added to 200 nmol of acetyl coenzyme A 
and either 0.1 (R3327 rat prostate cells) or 0.5 (T cells) .mu.Ci of 
[.sub.14 C]chloramphenicol (Amersham, Arlington Heights, Ill.). The 
reaction mixture is incubated at 37.degree. C. for 16 h. The acetylated 
and unacetylated chloramphenicol species are extracted with cold ethyl 
acetate and resolved on silica thin-layer chromatography plates with 95:5 
(vol/vol) chloroform-methanol solvent. The radiolabeled products are 
visualized by autoradiography. 
For Southern analysis, chromosomal DNA is extracted from cells by the 
procedure described by Hirt (Hirt, B., J. Mol. Biol. 126:275-288, 1967). 
After digestion with appropriate restriction enzymes. 5 .mu.g of DNA is 
loaded onto a 1% agarose gel, electrophoresed, and transferred to Hybond 
N+ (Amersham) nylon membrane. The membranes are hybridized with a 0.685-kb 
IL-2 gene fragment at 65.degree. C. in rapid hybridization buffer with DNA 
fragments labeled with .sup.32 P by the random priming method (Megaprime 
DNA labeling kit: Amersham) and washed according to the manufacturer's 
instructions. Autoradiograms of these filters are exposed on X-ray film 
(type XAR: Eastman Kodak Co.) with intensifying screens at 70.degree. C. 
for 1 to 4 days. 
The above-described DNA/liposome/AAV delivery system is useful for 
delivering a construct in single copy to the genome of a cell in that it 
allows the viral coat and capsid to be replaced by liposomes, yet includes 
sufficient viral elements to allow for stable inheritance via 
recombination of the construct with the host cell genome. Delivery of DNA 
by this means does not require interaction of the delivery vehicle with 
any specific cell surface receptor. 
A construct according to the invention, containing a CD2 COD to confer 
position independent low level expression of the associated IL-2 gene, 
when delivered to target cells, e.g., T-cells or tumor cells, may be 
tested for delivery and expression of the associated gene using the 
above-described procedures. The transfected cells may be used to modulate 
the cellular immune response in cancer and AIDS where a non-physiological 
level of a transgene product is desired to effect such modulation. 
EXAMPLE IX 
Also contemplated within the invention is the combination of a chromatin 
opening domain with a heterologous enhancer. A heterologous COD/enhancer 
combination allows one of skill in the art to choose a combination which 
will confer a desired level of transgene expression, i.e., that is not 
achievable using a homologous COD/enhancer combination. The heterologous 
COD/enhancer combination will achieve a level of transgene expression that 
is either less than or better than the level of transgene expression 
achieved using a homologous COD/enhancer combination. As used herein, 
"better than" or "less than" means at least 10% or preferably 20-25% 
different from the level of expression of a homologous COD/enhancer 
combination. 
Heterologous enhancers useful according to this aspect of the invention 
include but are not limited to the human Cytomegalovirus (CMV) enhancer, 
the .alpha.-globin 40 kb enhancer, SV40 enhancers, adenovirus enhancers, 
immunoglobulin enhancers, T cell receptor enhancers. In addition, the 
enhancer corresponding to the promoter and/or transgene present in the 
construct are useful according to the invention. 
Therefore, one construct according to the invention may include the 
.beta.-globin HS3 chromatin opening domain in combination with a 
heterologous enhancer such as the CMV enhancer or the .beta.-globin 
enhancer. Thus, where the transgene is the .beta.-globin gene, the HS3 
chromatin opening domain in combination with a heterologous enhancer will 
allow for increased expression of the .beta.-globin transgene in a tissue 
specific and position independent manner, but that level of expression 
will be either greater than or less than the level of expression of the 
transgene in combination with the complete .beta.-globin LCR. The 
chromatin opening domain/heterologous enhancer combination may be tested 
according to procedures described herein for expression of the 
.beta.-globin gene in combination with the .beta.-globin chromatin opening 
domain. 
EXAMPLE X 
The invention also encompasses treatment of Gaucher's disease. Gaucher's 
disease stems from one of two different genetic mutations. Gaucher's type 
1 is a CGG.fwdarw.CAG mutation, which results in an Arg.fwdarw.Gln 
substitution at position 119 of the .beta.-glucocerebrosidase polypeptide 
(Graves, DNA 7:521, 1988). Gaucher's type 2 is a CTG.fwdarw.CCG mutation, 
which results in a Leu.fwdarw.Pro substitution at position 444 of the 
.beta.-glucocerebrosidase polypeptide (Tsuji, NEJM 316:570, 1987). The 
presence of a .beta.-glucocerebrosidase gene encoding a wild type 
polypeptide is believed to substantially correct Gaucher's disease. 
Therefore, another construct according to the invention is one containing 
the chromatin opening domain of the macrophage-specific lysozyme gene LCR 
or the chromatin opening domain of the human lysozyme locus control region 
(see Bonifer et al., 1990, Euro. Mol. Biol. Org. Jour. 9;2843; and Bonifer 
et al., 1994, Nucleic Acids Research 22:4202-4210). These chromatin 
opening domains may be identified according to the procedures used, as 
described herein, for localization of the .beta.-globin chromatin opening 
domain. That is, the macrophage-specific lysozyme chromatin opening domain 
will retain the tissue-specificity of the full-length lysozyme LCR, and 
thus will direct transgene expression primarily in macrophages, and will 
also retain position-independent transgene expression of the full length 
LCR, but will not allow for expression of the transgene to the level 
conferred by the full length LCR. The reduced level of transgene 
expression conferred by the macrophage-specific lysozyme chromatin opening 
domain will be less than 60% of the level of expression of the transgene 
when associated with the full length corresponding LCR, will likely be 
less than 40%, and may be on the order of 10-25%. 
A construct containing the macrophage-specific lysozyme chromatin opening 
domain may also contain the lysozyme gene promoter and the 
.beta.-glucocerebrosidase transgene (Horowitz et al., 1989, Genomics 
4:87-96). This construct is made as follows. 
The human .beta.-glucocerebrosidase gene is carried, as disclosed in 
Horowitz et al., on a a 9722 base pair fragment extending from a BamHI 
site in exon 1 to an EcoRV site 3' to polyadenylation site. This fragment 
contains 11 exons and all intervening sequences, with translational start 
in exon 2. The chicken lysozyme LCR and promoter is carried on an 11.8 kb 
XhoI-SacI fragment from pIII.lyx construct as described in Bonifer et al., 
1990 supra. This 11.8 kb fragment may be shortened to determine the 
location of its chromatin opening domain, as described herein, and the 
shortened fragment ligated to the human .beta.-glucocerebrosidase gene. 
This construct may be used to treat Gaucher's disease by introducing the 
construct into macrophages, as described in Immunology and Cell Biology, 
1993, Vol. 71, pages 75-78 and introducing the transfected macrophages 
into a patient afflicted with Gaucher's disease. Expression of the wild 
type transgene in a patient afflicted with Gaucher's disease should result 
in correction of the diseased state. 
EXAMPLE XI 
The invention encompasses treatment of the genetic blood disorder X-linked 
.gamma.-globulinemia. This disease may be treated by introducing a 
construct including a chromatin opening domain into pre-B cells, and 
subsequently introducing the genetically altered pre-B cells into a 
patient afflicted with the disorder. 
Therefore, another construct according to the invention is one containing 
the chromatin opening domain of the class II major histocompatibility 
complex (MHC) gene LCR (Carson & Wiles, 1993, Nucleic Acids Research 
21:2065-2072). The chromatin opening domain of this LCR may be identified 
according to the procedures used, as described herein, for localization of 
the .beta.-globin LCR chromatin opening domain. That is, the class II MHC 
chromatin opening domain will retain the tissue-specificity of the 
full-length class II MHC LCR, and thus will direct transgene expression 
primarily in B or pre-B cells, and will also retain position-independent 
transgene expression of the full length LCR, but will not allow for 
expression of the transgene to the level conferred by the full length LCR. 
The reduced level of transgene expression conferred by the class II MHC 
chromatin opening domain will be less than 60% of the level of expression 
of the transgene when associated with the full length corresponding LCR, 
will likely be less than 40%, and may be on the order of 10-25%. The 
chromatin opening domain from the class II MHC will be larger than a 
fragment containing a deletion of three of the five DNase hypersensitive 
sites mapped in the MHCII LCR, as disclosed in Carson & Wiles, supra. 
A construct containing the class II MHC chromatin opening domain may also 
contain the Bruton's kinase transgene (Vetrie et al., 1993, Nature 
361:226-233). This construct may be made utilizing the following DNA 
fragments, which are cloned together using procedures well-known in the 
art. The Bruton's Tyrosine Kinase human gene is carried on a 2.1 kb 
fragment delineated by the PvuI site at position (+33) and the HindIII 
site at position (+2126). A region of the MHC II locus that has been found 
to confer tissue specific, position independent gene expression on the 
murine I-Ea gene lies in a 25 kb region of 5' flanking sequences. This 25 
kb fragment is delineated by an MluI site and a PvuI site from cosmid 32.1 
(see Carson & Wiles), and can be shortened and tested for chromatin 
opening domain activity as described herein. Finally, the construct will 
also encode a splice site and poly A tail, which may include portions of 
the human B globin locus splice and poly A signals; i.e., a BamHI-XbaI 2.8 
kb 3' splice/poly A flankling sequence containing exon 2--IVSII--exon 
3--polyA sequences. 
The above-described construct may be used to treat X-linked 
.gamma.-globulinemia by introducing the construct into pre-B cells, as 
described in Martensson et al., Eur. Jour. Immunol. 1987, 17:1499; Okabe 
et al., Eur. Jour. Immunol. 1992, 22:37; and Banerji et al., Cell 33:729, 
1983, and administering the transfected pre-B cells into a patient 
afflicted with X-linked .gamma.-globulinemia. 
EXAMPLE XII 
Another construct according to the invention is a reporter construct for 
testing of chromatin opening domains of the invention. This construct 
contains the .beta.-galactosidase reporter gene driven by the mouse heat 
shock promoter 68 (hsp 68) and the .beta.-globin HS3 COD. The construct is 
then used to make a transgenic mouse. A transgenic mouse containing a 
single copy of the reporter construct is then tested for position 
independent reporter gene expression. The presence of a chromatin opening 
domain will be indicated by position independent, copy number dependent 
reporter gene expression that does not rise to the level of reporter gene 
expression obtained using a fully functional LCR. 
Other chromatin opening domains may be tested according to the invention 
and using the above-described reporter construct. For example, a candidate 
LCR is provided and subcloned. LCR subregions are then inserted into the 
reporter construct and tested in vivo for tissue-specific, position 
independent expression. Where such expression is found, a chromatin 
opening domain is indicated, but only if the level of transgene expression 
does not rise to the level of expression of the transgene when associated 
with the corresponding full length LCR. That is, the level of expression 
of a transgene when associated with a COD will be on the order of 10-25% 
of the level of transgene expression with the full length LCR. 
OTHER EMBODIMENTS 
Other embodiments will be evident to those of skill in the art. It should 
be understood that the foregoing detailed description is provided for 
clarity only and is merely exemplary. The spirit and scope of the present 
invention are not limited to the above examples, but are encompassed by 
the following claims. 
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