U.S. patent application number 14/537312 was filed with the patent office on 2015-05-21 for non-human animals having a humanized b-cell activating factor gene.
The applicant listed for this patent is Regeneron Pharmaceuticals, Inc.. Invention is credited to Cagan Gurer, Lynn Macdonald, John McWhirter, Andrew J. Murphy.
Application Number | 20150143558 14/537312 |
Document ID | / |
Family ID | 51982794 |
Filed Date | 2015-05-21 |
United States Patent
Application |
20150143558 |
Kind Code |
A1 |
McWhirter; John ; et
al. |
May 21, 2015 |
NON-HUMAN ANIMALS HAVING A HUMANIZED B-CELL ACTIVATING FACTOR
GENE
Abstract
Non-human animals, cells, methods and compositions for making
and using the same are provided, wherein the non-human animals and
cells comprise a humanized B-cell activating factor gene. Non-human
animals and cells that express a human or humanized B-cell
activating factor protein from an endogenous B-cell activating
factor locus are described.
Inventors: |
McWhirter; John; (Tarrytown,
NY) ; Gurer; Cagan; (Valhalla, NY) ;
Macdonald; Lynn; (White Plains, NY) ; Murphy; Andrew
J.; (Croton-on-Hudson, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Regeneron Pharmaceuticals, Inc. |
Tarrytown |
NY |
US |
|
|
Family ID: |
51982794 |
Appl. No.: |
14/537312 |
Filed: |
November 10, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61905983 |
Nov 19, 2013 |
|
|
|
Current U.S.
Class: |
800/9 ; 424/93.7;
435/29; 435/354; 435/7.24; 435/7.92; 800/21 |
Current CPC
Class: |
A01K 67/0278 20130101;
A01K 2227/105 20130101; A01K 2207/12 20130101; A01K 2267/03
20130101; A01K 2207/15 20130101; G01N 2500/10 20130101; G01N
33/5088 20130101; C07K 2319/00 20130101; C07K 14/70575 20130101;
A01K 2217/072 20130101; A01K 2217/07 20130101; A01K 2267/0381
20130101; A01K 2267/0325 20130101; A01K 2267/0387 20130101 |
Class at
Publication: |
800/9 ; 435/354;
800/21; 424/93.7; 435/29; 435/7.24; 435/7.92 |
International
Class: |
A01K 67/027 20060101
A01K067/027; G01N 33/50 20060101 G01N033/50; C12N 15/85 20060101
C12N015/85 |
Claims
1. A non-human animal expressing a Baff polypeptide comprising the
extracellular portion of a human BAFF protein linked to the
intracellular portion of a Baff protein of the non-human
animal.
2. The non-human animal of claim 1, wherein the extracellular
portion of a human BAFF protein is encoded by exons 3 to 6 of a
human BAFF gene.
3.-6. (canceled)
7. The non-human animal of claim 2, wherein exons 3 to 6 of the
human BAFF gene are at least 90% identical with exons 3 to 6 of a
human BAFF gene that appears in Table 3.
8. (canceled)
9. The non-human animal of claim 1, wherein the non-human animal
does not detectably express a full-length endogenous Baff
protein.
10. The non-human animal of claim 1, wherein the Baff polypeptide
is expressed from a humanized Baff gene at an endogenous Baff
locus.
11. The non-human animal of claim 10, wherein the humanized Baff
gene comprises at least one non-human Baff exon selected from the
group consisting of exon 1, exon 2, and full or partial exon 7, of
a non-human Baff gene.
12.-13. (canceled)
14. A non-human animal comprising a humanized Baff gene that
comprises one or more exons of a human BAFF gene operably linked to
a Baff promoter.
15. The non-human animal of claim 14, wherein the Baff promoter is
a non-human animal Baff promoter.
16. The non-human animal of claim 14, wherein the Baff promoter is
a human BAFF promoter.
17. The non-human animal of claim 14, wherein the humanized Baff
gene comprises exons 3 to 6 of a human BAFF gene.
18.-21. (canceled)
22. The non-human animal of claim 17, wherein exons 3 to 6 of the
human BAFF gene are at least 90% identical with exons 3 to 6 of a
human BAFF gene that appears in Table 3.
23. (canceled)
24. The non-human animal of claim 14, wherein the humanized Baff
gene comprises a non-human Baff exon selected from the group
consisting of exon 1, exon 2, and full or partial exon 7, of a
non-human Baff gene.
25.-31. (canceled)
32. An isolated cell or tissue of the non-human animal of claim
14.
33.-38. (canceled)
39. A method of making a non-human animal that expresses a Baff
protein from an endogenous Baff locus, wherein the Baff protein
comprises a human sequence, the method comprising the steps of: (a)
targeting an endogenous Baff gene at an endogenous Baff locus in a
non-human embryonic stem (ES) cell with a genomic fragment
comprising a human nucleotide sequence that encodes a human BAFF
protein in whole or in part; (b) obtaining a modified non-human ES
cell comprising a humanized Baff gene that comprises the human
nucleotide sequence of (a) at the endogenous Baff locus; and, (c)
creating a non-human animal using the modified ES cell of (b).
40. The method of claim 39, wherein the human nucleotide sequence
comprises exons 3 to 6 of a human BAFF gene.
41. The method of claim 39, wherein the human nucleotide sequence
encodes amino acid residues 142 to 285 of a human BAFF protein.
42. The method of claim 39, wherein the non-human animal is a mouse
or rat.
43.-45. (canceled)
46. A method of engrafting human cells into a mouse, the method
comprising steps of: (a) providing a mouse whose genome comprises a
humanized Baff gene that encodes the extracellular portion of a
human BAFF protein linked to the intracellular portion of a mouse
Baff protein; and (b) transplanting one or more human cells into
the mouse.
47. (canceled)
48. The method of claim 46, further comprising a step of: (c)
assaying engraftment of the one or more human cells in the mouse,
wherein the step of assaying comprises comparing the engraftment of
the one or more human cells to the engraftment in one or more
wild-type mice or in one or more mice whose genome does not
comprise a humanized Baff gene that encodes the extracellular
portion of a human BAFF protein linked to the intracellular portion
of a mouse Baff protein.
49.-50. (canceled)
51. The method of claim 46, wherein the human cells are
hematopoietic stem cells.
52. The method of claim 46, wherein the human cells are
transplanted intravenously, intraperitoneally, or
subcutaneously.
53.-54. (canceled)
55. The non-human animal of claim 1, wherein the non-human animal
is a rodent.
56. The non-human animal of claim 14, wherein the non-human animal
is a rodent.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of priority of U.S.
Provisional Application No. 61/905,983, filed Nov. 19, 2013, the
entire content of which is incorporated herein by reference.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
[0002] The Sequence Listing in the ASCII text file, named as
31015.sub.--6800_SEQ.txt of 22 KB bytes, created on Nov. 5, 2014,
and submitted to the United States Patent and Trademark Office via
EFS-Web, is incorporated herein by reference.
BACKGROUND
[0003] Autoimmunity results when an organism's natural mechanisms
for preventing its immune system from attacking its own cells and
tissues break down. Diseases, disorders and conditions caused by
breakdown, and by the aberrant self-directed immune responses that
result, are referred to as autoimmune diseases. Notable examples of
autoimmune diseases, disorders and conditions include diabetes
mellitus, systemic lupus erythematosus (SLE), rheumatoid arthritis
(RA) and some allergies. Autoimmune diseases are estimated to be
among the ten leading causes of death. Investment in the
development of therapies for autoimmune diseases is in the
multi-billion dollar range and critical in vivo systems to test,
develop and validate candidate therapeutics are necessary to ensure
treatment safety and effectiveness. Further, such in vivo systems
are necessary in determining if new treatments can sustain long
term improvement in patients and, perhaps, can even provide cures
for many diseases that remain unaddressed. Such in vivo systems
also provide a source for assays in human hematopoietic and immune
system related functions in vivo, identification of novel therapies
and vaccines.
SUMMARY OF INVENTION
[0004] The present invention encompasses the recognition that it is
desirable to engineer non-human animals to provide improved in vivo
autoimmune disease systems to permit the testing, development and
validation of new and existing candidate therapeutics. The present
invention also encompasses the recognition that it is desirable to
engineer non-human animals to permit improved activation and
survival of human lymphocytes (e.g., B cells) post-immunization and
post-engraftment of human hematopoietic stem cells or B cells from
human donors. The present invention also encompasses the
recognition that non-human animals having a humanized Baff gene
and/or otherwise expressing, containing, or producing a human or
humanized Baff protein are desirable, for example for use in
engraftment of human hematopoietic stem cells or B cells from human
donors.
[0005] In some embodiments, a non-human animal of the present
invention expresses a Baff polypeptide comprising the extracellular
portion of a human BAFF protein linked to the intracellular portion
of a mouse Baff protein.
[0006] In some embodiments, an extracellular portion of a human
BAFF protein is encoded by exons 3 to 6 of a human BAFF gene.
[0007] In some embodiments, exons 3 to 6 of a human BAFF gene are
at least 50%, at least 55%, at least 60%, at least 65%, at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, or at least 98% identical with exons 3 to 6 of a human
BAFF gene that appears in Table 3. In some embodiments, exons 3 to
6 of a human BAFF gene are 100% identical with exons 3 to 6 of a
human BAFF gene that appears in Table 3.
[0008] In some embodiments, a non-human animal of the present
invention does not detectably express a full-length endogenous Baff
protein. In some embodiments, the non-human animal is a rodent and
does not detectably express a full-length rodent Baff protein. In
some embodiments, the non-human animal is a mouse and does not
detectably express a full-length mouse Baff protein whose sequence
appears in Table 3.
[0009] In some embodiments, a Baff polypeptide of the present
invention is expressed from a genetically modified Baff gene at an
endogenous non-human Baff locus. In some certain embodiments, a
genetically modified Baff gene comprises a non-human Baff exon 1.
In some certain embodiments, a genetically modified Baff gene
comprises a non-human Baff exon 2. In some certain embodiments, a
genetically modified Baff gene comprises a non-human Baff exon 7 in
whole or in part. In some certain embodiments, a genetically
modified Baff gene comprises a non-human Baff exon 1 and exon 2. In
some certain embodiments, a genetically modified Baff gene
comprises a non-human Baff exon 1, a non-human Baff exon 2, a
non-human Baff exon 7 in whole or in part, or a combination
thereof. In various embodiments, a non-human Baff exon 7 in part
comprises a non-human Baff 3'untranslated region (UTR) and a
non-human Baff polyadenylation signal.
[0010] In some embodiments, the present invention provides a
non-human animal comprising a genetically modified Baff gene that
comprises one or more exons of a human BAFF gene (i.e., a humanized
Baff gene) operably linked to a Baff promoter. In some embodiments,
a Baff promoter of the present invention is a non-human Baff
promoter. In some embodiments, a BAFF promoter of the present
invention is a human Baff promoter.
[0011] In some embodiments, a humanized Baff gene of the present
invention comprises exons 3 to 6 of a human BAFF gene. In some
certain embodiments, a humanized Baff gene further comprises a
non-human Baff exon 1. In some certain embodiments, a humanized
Baff gene further comprises a non-human Baff exon 2. In some
certain embodiments, a humanized BAFF gene further comprises a
non-human Baff exon 7 in whole or in part. In some certain
embodiments, a humanized Baff gene comprises a non-human Baff exon
1, exon 2 and a non-human Baff exon 7 in whole or in part. In
various embodiments, a non-human Baff exon 7 in part comprises a
non-human Baff 3'untranslated region (UTR) and a non-human Baff
polyadenylation signal.
[0012] In some embodiments, exons 3 to 6 of a human BAFF gene are
at least 50%, at least 55%, at least 60%, at least 65%, at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, or at least 98% identical with exons 3 to 6 of a human
BAFF gene that appears in Table 3. In some embodiments, exons 3 to
6 of a human BAFF gene are 100% identical with exons 3 to 6 of a
human BAFF gene that appears in Table 3.
[0013] In various embodiments, a non-human animal of the present
invention is a rodent. In some certain embodiments, a rodent of the
present invention is selected from a mouse or a rat.
[0014] In some embodiments, the present invention provides a
humanized Baff locus (or gene) comprising one or more exons of a
non-human Baff gene operably linked to one or more exons of a human
BAFF gene.
[0015] In some embodiments, a humanized Baff locus (or gene) of the
present invention comprises non-human Baff exons 1 and 2 operably
linked to human BAFF exons 3 to 6. In some certain embodiments, a
humanized Baff locus (or gene) further comprises 5' and 3'
non-human untranslated regions (UTRs) flanking a non-human Baff
exon 1 and a human BAFF exon 6.
[0016] In some embodiments, the present invention provides a Baff
polypeptide encoded by a humanized Baff locus, or gene, as
described herein.
[0017] In some embodiments, the present invention provides a cell
or tissue isolated from a non-human animal as described herein. In
some embodiments, a cell is selected from an astrocyte, dendritic
cell, lymphocyte (e.g., a B or T cell), monocyte, neutrophils and a
stromal cell. In some embodiments, a tissue is selected from
adipose, bladder, brain, breast, bone marrow, eye, heart,
intestine, kidney, liver, lung, lymph node, muscle, pancreas,
plasma, serum, skin, spleen, stomach, thymus, testis, ovum, and/or
a combination thereof.
[0018] In some embodiments, the present invention provides an
isolated non-human (e.g., rodent) cell or tissue whose genome
includes a Baff gene (or locus) comprising one or more exons of a
non-human Baff gene operably linked to one or more exons of a human
BAFF gene. In some certain embodiments, the present invention
provides an isolated non-human (e.g., rodent) cell or tissue whose
genome includes a Baff gene (or locus) comprising non-human Baff
exons 1 and 2 operably linked to human BAFF exons 3 to 6, wherein
the Baff gene (or locus) further comprises 5' and 3' non-human
untranslated regions (UTRs) flanking the non-human Baff exon 1 and
the human BAFF exon 6. In some embodiments, a Baff gene (or locus)
comprises a sequence that encodes a BAFF polypeptide that comprises
residues 142 to 285 of a human BAFF protein.
[0019] In some embodiments, the present invention provides a
non-human embryonic stem (ES) cell whose genome comprises a Baff
gene (or locus) as described herein. In some certain embodiments,
the ES cell comprises a Baff gene that encodes the extracellular
portion of a human BAFF protein linked to the intracellular portion
of a mouse Baff protein. In some certain embodiments, the ES cell
comprises a Baff gene that comprises exons 3 to 6 of a human BAFF
gene. In some certain embodiments, the ES cell is a rodent ES cell.
In some embodiments, a non-human ES cell of the present invention
is a mouse or rat ES cell.
[0020] In some embodiments, the present invention provides the use
of a non-human embryonic stem cell as described herein to make a
non-human animal. In some certain embodiments, a non-human
embryonic stem cell is murine and is used to make a mouse
comprising a Baff gene as described herein.
[0021] In some embodiments, the present invention provides a
non-human embryo comprising, made from, obtained from, or generated
from a non-human embryonic stem cell comprises a Baff gene as
described herein. In some embodiments, a non-human embryo of the
present invention is a rodent embryo. In some embodiments, a rodent
embryo as described herein is a mouse or rat embryo.
[0022] In some embodiments, the present invention provides a method
of making a non-human animal that expresses a Baff protein from a
humanized Baff gene at an endogenous Baff locus, wherein the Baff
protein comprises a human sequence, the method comprising the steps
of targeting an endogenous Baff gene (or locus) in a non-human
embryonic stem (ES) cell with a genomic fragment comprising a human
nucleotide sequence that encodes a human BAFF protein in whole or
in part, obtaining a modified non-human embryonic stem (ES) cell
comprising a humanized Baff gene at an endogenous Baff locus that
comprises said human sequence, and creating a non-human animal
using said modified embryonic stem (ES) cell.
[0023] In some embodiments, said human nucleotide sequence
comprises exons 3 to 6 of a human BAFF gene. In some embodiments,
said human nucleotide sequence comprises exons 3 to 6 of a human
BAFF gene that are at least 50%, at least 55%, at least 60%, at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least 95%, or at least 98% identical with exons 3
to 6 of a human BAFF gene that appears in Table 3. In some certain
embodiments, said human nucleotide sequence comprises exons 3 to 6
of a human BAFF gene that are 100% identical with exons 3 to 6 of a
human BAFF gene that appears in Table 3.
[0024] In some embodiments, said human nucleotide sequence encodes
amino acid residues 142 to 285 of a human BAFF protein. In some
embodiments, said human nucleotide sequence encodes amino acid
residues 142-295 of a human BAFF protein that are at least 50%, at
least 55%, at least 60%, at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, at least 95%, or at least
98% identical with amino acid residues 142-295 of a human BAFF
protein that appears in Table 3. In some certain embodiments, said
human nucleotide sequence encodes amino acid residues 142-295 of a
human BAFF protein that are 100% identical with amino acid residues
142-295 of a human BAFF protein that appears in Table 3.
[0025] In some embodiments, the present invention provides a mouse
or a rat made by, or obtained (or obtainable) from, a method as
described herein. In some certain embodiments, a mouse or a rat
made by, or obtained (or obtainable) from, a method as described
herein does not detectably express a full-length endogenous (e.g.,
mouse or rat) Baff protein.
[0026] In some embodiments, the present invention provides a method
of providing a mouse whose genome includes a Baff gene that encodes
the extracellular portion of a human BAFF protein linked to the
intracellular portion of a mouse Baff protein, the method
comprising modifying the genome of a mouse so that it comprises a
Baff gene that encodes the extracellular portion of a human BAFF
protein linked to the intracellular portion of a mouse Baff protein
thereby providing said mouse. In some embodiments, a Baff gene is a
Baff gene as described herein. In some embodiments, a Baff gene is
one that encodes a protein whose sequence reflects a humanized Baff
protein that appears in Table 3. In some certain embodiments, a
Baff gene comprises exons 3 to 6 a human BAFF gene.
[0027] In various embodiments, a humanized Baff gene of the present
invention comprises exons 3, 4, 5 and 6 of a human BAFF gene. In
various embodiments, an extracellular portion of a humanized Baff
protein of the present invention comprises amino acids
corresponding to residues 142-295 of a human BAFF protein that
appears in Table 3. In some certain embodiments, a humanized Baff
protein of the present invention comprises a sequence of a
humanized Baff protein that appears in Table 3. In various
embodiments, a humanized Baff gene of the present invention is
operably linked to a mouse Baff promoter.
[0028] In some embodiments, the present invention provides a method
of engrafting human cells into a mouse, the method comprising steps
of providing a mouse whose genome comprises a Baff gene that
encodes the extracellular portion of a human BAFF protein linked to
the intracellular portion of a mouse Baff protein (as described
herein), and transplanting one or more human cells into the mouse.
In some certain embodiments, the method further comprises a step of
assaying engraftment of the one or more human cells in the mouse.
In some certain embodiments, the step of assaying comprises
comparing the engraftment of the one or more human cells to the
engraftment in one or more wild-type mice or in one or more mice
whose genome does not comprise a Baff gene that encodes the
extracellular portion of a human BAFF protein linked to the
intracellular portion of a mouse Baff protein.
[0029] In some certain embodiments, the human cells are
hematopoietic stem cells. In some certain embodiments, the human
cells are human B cells.
[0030] In some embodiments, the human cells are transplanted
intravenously. In some embodiments, the human cells are
transplanted intraperitoneally. In some embodiments, the human
cells are transplanted subcutaneously.
[0031] In some embodiments, the present invention provides methods
for identification or validation of a drug or vaccine, the method
comprising the steps of delivering a drug or vaccine to a non-human
animal as described herein, and monitoring one or more of the
immune response to the drug or vaccine, the safety profile of the
drug or vaccine, or the effect on a disease or condition. In some
embodiments, monitoring the safety profile includes determining if
the non-human animal exhibits a side effect or adverse reaction as
a result of delivering the drug or vaccine. In some embodiments, a
side effect or adverse reaction is selected from morbidity,
mortality, alteration in body weight, alteration of the level of
one or more enzymes (e.g., liver), alteration in the weight of one
or more organs, loss of function (e.g., sensory, motor, organ,
etc.), increased susceptibility to one or more diseases,
alterations to the genome of the non-human animal, increase or
decrease in food consumption and complications of one or more
diseases.
[0032] In some embodiments, the present invention provides use of a
non-human animal of the present invention in the development of a
drug or vaccine for use in medicine, such as use as a
medicament.
[0033] In various embodiments, non-human animals of the present
invention are rodents, preferably a mouse or a rat.
[0034] As used in this application, the terms "about" and
"approximately" are used as equivalents. Any numerals used in this
application with or without about/approximately are meant to cover
any normal fluctuations appreciated by one of ordinary skill in the
relevant art.
[0035] Other features, objects, and advantages of the present
invention are apparent in the detailed description that follows. It
should be understood, however, that the detailed description, while
indicating embodiments of the present invention, is given by way of
illustration only, not limitation. Various changes and
modifications within the scope of the invention will become
apparent to those skilled in the art from the detailed
description.
BRIEF DESCRIPTION OF THE DRAWING
[0036] The Drawing included herein, which is comprised of the
following Figures, is for illustration purposes only not for
limitation.
[0037] FIG. 1 shows a diagram, not to scale, of the genomic
organization of mouse and human B-cell Activating Factor (BAFF)
genes. Exons are numbered beneath each exon.
[0038] FIG. 2 shows a diagram, not to scale, of an exemplary method
for humanization of a non-human B-cell Activating Factor (Baff)
gene. Non-human sequences are shown as closed, black symbols. Human
sequences are shown in open, diagonal filled symbols. CM:
Chloramphenicol selection cassette. Hyg: hygromycin selection
cassette. SDC NEO: self-deleting neomycin selection cassette. Spec:
spectinomycin selection cassette. Frt: Flp recombinase target
recognition site sequence. LoxP: Cre recombinase target recognition
site sequence. Restriction enzyme recognition sites are indicated
(e.g., AsiSI, I-CeuI, etc.).
DEFINITIONS
[0039] This invention is not limited to particular methods, and
experimental conditions described, as such methods and conditions
may vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments
only, and is not intended to be limiting, since the scope of the
present invention is defined by the claims.
[0040] Unless defined otherwise, all terms and phrases used herein
include the meanings that the terms and phrases have attained in
the art, unless the contrary is clearly indicated or clearly
apparent from the context in which the term or phrase is used.
Although any methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present invention, particular methods and materials are now
described. All publications mentioned are hereby incorporated by
reference.
[0041] The term "approximately" as applied herein to one or more
values of interest, refers to a value that is similar to a stated
reference value. In certain embodiments, the term "approximately"
or "about" refers to a range of values that fall within 25%, 20%,
19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%,
5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or
less than) of the stated reference value unless otherwise stated or
otherwise evident from the context (except where such number would
exceed 100% of a possible value).
[0042] The term "biologically active" as used herein refers to a
characteristic of any agent that has activity in a biological
system, in vitro or in vivo (e.g., in an organism). For instance,
an agent that, when present in an organism, has a biological effect
within that organism, is considered to be biologically active. In
particular embodiments, where a protein or polypeptide is
biologically active, a portion of that protein or polypeptide that
shares at least one biological activity of the protein or
polypeptide is typically referred to as a "biologically active"
portion.
[0043] The term "comparable", as used herein, refers to two or more
agents, entities, situations, sets of conditions, etc. that may not
be identical to one another but that are sufficiently similar to
permit comparison there between so that conclusions may reasonably
be drawn based on differences or similarities observed. Those of
ordinary skill in the art will understand, in context, what degree
of identity is required in any given circumstance for two or more
such agents, entities, situations, sets of conditions, etc. to be
considered comparable.
[0044] The term "conservative" as used herein to describe a
conservative amino acid substitution refers to substitution of an
amino acid residue by another amino acid residue having a side
chain R group with similar chemical properties (e.g., charge or
hydrophobicity). In general, a conservative amino acid substitution
will not substantially change the functional properties of interest
of a protein, for example, the ability of a receptor to bind to a
ligand. Examples of groups of amino acids that have side chains
with similar chemical properties include aliphatic side chains such
as glycine, alanine, valine, leucine, and isoleucine;
aliphatic-hydroxyl side chains such as serine and threonine;
amide-containing side chains such as asparagine and glutamine;
aromatic side chains such as phenylalanine, tyrosine, and
tryptophan; basic side chains such as lysine, arginine, and
histidine; acidic side chains such as aspartic acid and glutamic
acid; and, sulfur-containing side chains such as cysteine and
methionine. Conservative amino acids substitution groups include,
for example, valine/leucine/isoleucine, phenylalanine/tyrosine,
lysine/arginine, alanine/valine, glutamate/aspartate, and
asparagine/glutamine. In some embodiments, a conservative amino
acid substitution can be substitution of any native residue in a
protein with alanine, as used in, for example, alanine scanning
mutagenesis. In some embodiments, a conservative substitution is
one that that has a positive value in the PAM250 log-likelihood
matrix disclosed in Gonnet et al. (1992) Exhaustive Matching of the
Entire Protein Sequence Database, Science 256:1443-45, hereby
incorporated by reference. In some embodiments, a substitution is
deemed to be "moderately conservative" if it has a nonnegative
value in the PAM250 log-likelihood matrix.
[0045] The term "disruption" as used herein refers to the result of
an event that interrupts (e.g., via homologous recombination) a
DNA. In some embodiments, a disruption may achieve or represent a
deletion, insertion, inversion, modification, replacement,
substitution, or any combination thereof, of a DNA sequence(s). In
some embodiments, a disruption may achieve or represent
introduction of a mutation, such as a missense, nonsense, or
frame-shift mutation, or any combination thereof, in a coding
sequence(s) in DNA. In some embodiments, a disruption may occur in
a gene or gene locus endogenous to a cell. In some embodiments,
insertions may include the insertion of entire genes or fragments
of genes, e.g. exons, in to an endogenous site in a cell or genome.
In some embodiments, insertions may introduce sequences that are of
an origin other than that of an endogenous sequence into which they
are inserted. In some embodiments, a disruption may increase
expression and/or activity of a gene or gene product (e.g., of a
protein encoded by a gene). In some embodiments, a disruption may
decrease expression and/or activity of a gene or gene product. In
some embodiments, a disruption may alter sequence of a gene or gene
product (e.g., an encoded protein). In some embodiments, a
disruption may truncate or fragment a gene or gene product (e.g.,
an encoded protein). In some embodiments, a disruption may extend a
gene or gene product; in some such embodiments, a disruption may
achieve assembly of a fusion protein. In some embodiments, a
disruption may affect level but not activity of a gene or gene
product. In some embodiments, a disruption may affect activity but
not level of a gene or gene product. In some embodiments, a
disruption may have no significant effect on level of a gene or
gene product. In some embodiments, a disruption may have no
significant effect on activity of a gene or gene product. In some
embodiments, a disruption may have no significant effect on either
level or activity of a gene or gene product.
[0046] The phrase "endogenous locus" or "endogenous gene" as used
herein refers to a genetic locus found in a parent or reference
organism prior to introduction of a disruption (e.g., insertion,
inversion, modification, replacement, substitution, or a
combination thereof as described herein). In some embodiments, an
endogenous locus has a sequence found in nature. In some
embodiments, an endogenous locus is wild type. In some embodiments,
a reference organism that contains an endogenous locus as described
herein is a wild-type organism. In some embodiments, a reference
organism that contains an endogenous locus as described herein is
an engineered organism. In some embodiments, a reference organism
that contains an endogenous locus as described herein is a
laboratory-bred organism (whether wild-type or engineered).
[0047] The phrase "endogenous promoter" refers to a promoter that
is naturally associated, e.g., in a wild-type organism, with an
endogenous gene.
[0048] The term "heterologous" as used herein refers to an agent or
entity from a different source. For example, when used in reference
to a polypeptide, gene, or gene product or present in a particular
cell or organism, the term clarifies that the relevant polypeptide,
gene, or gene product 1) was engineered by the hand of man; 2) was
introduced into the cell or organism (or a precursor thereof)
through the hand of man (e.g., via genetic engineering); and/or 3)
is not naturally produced by or present in the relevant cell or
organism (e.g., the relevant cell type or organism type).
[0049] The term "host cell", as used herein, refers to a cell into
which a heterologous (e.g., exogenous) nucleic acid or protein has
been introduced. Persons of skill upon reading this disclosure will
understand that such terms refer not only to a particular subject
cell, but also is used to refer to progeny of that cell. Because
certain modifications may occur in succeeding generations due to
either mutation or environmental influences, such progeny may not,
in fact, be identical to the parent cell, but are still understood
by those skilled in the art to be included within the scope of the
term "host cell" as used herein. In some embodiments, a host cell
is or comprises a prokaryotic or eukaryotic cell. In general, a
host cell is any cell that is suitable for receiving and/or
producing a heterologous nucleic acid or protein, regardless of the
Kingdom of life to which the cell is designated. Exemplary cells
that may be utilized as host cells in accordance with the present
disclosure include those of prokaryotes and eukaryotes (single-cell
or multiple-cell), bacterial cells (e.g., strains of E. coli,
Bacillus spp., Streptomyces spp., etc.), mycobacteria cells, fungal
cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, P.
methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21,
baculovirus-infected insect cells, Trichoplusia ni, etc.),
non-human animal cells, human cells, or cell fusions such as, for
example, hybridomas or quadromas. In some embodiments, the cell is
a human, monkey, ape, hamster, rat, or mouse cell. In some
embodiments, the cell is eukaryotic and is selected from the
following cells: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS
(e.g., COS-7), retinal cell, Vero, CV1, kidney (e.g., HEK293, 293
EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205,
HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal),
CV-1, U937, 3T3, L cell, C127 cell, SP2/0, NS-0, MMT 060562,
Sertoli cell, BRL 3A cell, HT1080 cell, myeloma cell, tumor cell,
and a cell line derived from an aforementioned cell. In some
embodiments, the cell comprises one or more viral genes, e.g., a
retinal cell that expresses a viral gene (e.g., a PER.C6.TM. cell).
In some embodiments, a host cell is or comprises an isolated cell.
In some embodiments, a host cell is part of a tissue. In some
embodiments, a host cell is part of an organism.
[0050] The term "humanized", is used herein in accordance with its
art-understood meaning to refer to nucleic acids or proteins whose
structures (i.e., nucleotide or amino acid sequences) include
portions that correspond substantially or identically with versions
of the relevant nucleic acids or proteins that are found in nature
in non-human animals and that are distinguishable from
corresponding versions that are found in nature in humans, and also
include portions whose structures differ from those present in the
non-human-animal versions and instead correspond more closely with
comparable structures found in the human versions. In some
embodiments, a "humanized" gene is one that encodes a polypeptide
having substantially the amino acid sequence as that of a human
polypeptide (e.g., a human protein or portion thereof--e.g.,
characteristic portion thereof). To give but one example, in the
case of a membrane receptor, a "humanized" gene may encode a
polypeptide with an extracellular portion whose amino acid sequence
is identical or substantially identical to that of a human
extracellular portion, and whose remaining sequence is identical or
substantially identical to that of a non-human (e.g., mouse)
polypeptide. In some embodiments, a humanized gene comprises at
least a portion of an DNA sequence of a human gene. In some
embodiment, a humanized gene comprises an entire DNA sequence found
in a human gene. In some embodiments, a humanized protein has an
amino acid sequence that comprises a portion that appears in a
human protein. In some embodiments, a humanized protein has an
amino acid sequence whose entire sequence is found in a human
protein. In some embodiments (including, for example, some in which
a humanized protein has an amino acid sequence whose entire
sequence is found in a human protein), a humanized protein is
expressed from an endogenous locus of a non-human animal, which
endogenous locus corresponds to the homolog or ortholog of the
relevant human gene encoding the protein.
[0051] The term "identity" as used herein in connection with a
comparison of sequences, refers to identity as determined by any of
a number of different algorithms known in the art that can be used
to measure nucleotide and/or amino acid sequence identity. In some
embodiments, identities as described herein are determined using a
ClustalW v. 1.83 (slow) alignment employing an open gap penalty of
10.0, an extend gap penalty of 0.1, and using a Gonnet similarity
matrix (MACVECTOR.TM. 10.0.2, MacVector Inc., 2008). As used
herein, the term "identity" refers to the overall relatedness
between polymeric molecules, e.g., between nucleic acid molecules
(e.g., DNA molecules and/or RNA molecules) and/or between
polypeptide molecules. In some embodiments, polymeric molecules are
considered to be "substantially identical" to one another if their
sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. As will be
understood by those skilled in the art, a variety of algorithms are
available that permit comparison of sequences in order to determine
their degree of homology, including by permitting gaps of
designated length in one sequence relative to another when
considering which residues "correspond" to one another in different
sequences. Calculation of the percent identity between two nucleic
acid sequences, for example, can be performed by aligning the two
sequences for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second nucleic acid
sequences for optimal alignment and non-corresponding sequences can
be disregarded for comparison purposes). In certain embodiments,
the length of a sequence aligned for comparison purposes is at
least 30%, at least 40%, at least 50%, at least 60%, at least 70%,
at least 80%, at least 90%, at least 95%, or substantially 100% of
the length of the reference sequence. The nucleotides at
corresponding nucleotide positions are then compared. When a
position in the first sequence is occupied by the same nucleotide
as the corresponding position in the second sequence, then the
molecules are identical at that position. The percent identity
between the two sequences is a function of the number of identical
positions shared by the sequences, taking into account the number
of gaps, and the length of each gap, which needs to be introduced
for optimal alignment of the two sequences. Representative
algorithms and computer programs useful in determining the percent
identity between two nucleotide sequences include, for example, the
algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has
been incorporated into the ALIGN program (version 2.0) using a
PAM120 weight residue table, a gap length penalty of 12 and a gap
penalty of 4. The percent identity between two nucleotide sequences
can, alternatively, be determined for example using the GAP program
in the GCG software package using an NWSgapdna.CMP matrix.
[0052] The term "isolated", as used herein, refers to a substance
and/or entity that has been (1) separated from at least some of the
components with which it was associated when initially produced
(whether in nature and/or in an experimental setting), and/or (2)
designed, produced, prepared, and/or manufactured by the hand of
man. Isolated substances and/or entities may be separated from
about 10%, about 20%, about 30%, about 40%, about 50%, about 60%,
about 70%, about 80%, about 90%, about 91%, about 92%, about 93%,
about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,
or more than about 99% of the other components with which they were
initially associated. In some embodiments, isolated agents are
about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,
about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,
or more than about 99% pure. As used herein, a substance is "pure"
if it is substantially free of other components. In some
embodiments, as will be understood by those skilled in the art, a
substance may still be considered "isolated" or even "pure", after
having been combined with certain other components such as, for
example, one or more carriers or excipients (e.g., buffer, solvent,
water, etc.); in such embodiments, percent isolation or purity of
the substance is calculated without including such carriers or
excipients. To give but one example, in some embodiments, a
biological polymer such as a polypeptide or polynucleotide that
occurs in nature is considered to be "isolated" when, a) by virtue
of its origin or source of derivation is not associated with some
or all of the components that accompany it in its native state in
nature; b) it is substantially free of other polypeptides or
nucleic acids of the same species from the species that produces it
in nature; c) is expressed by or is otherwise in association with
components from a cell or other expression system that is not of
the species that produces it in nature. Thus, for instance, in some
embodiments, a polypeptide that is chemically synthesized or is
synthesized in a cellular system different from that which produces
it in nature is considered to be an "isolated" polypeptide.
Alternatively or additionally, in some embodiments, a polypeptide
that has been subjected to one or more purification techniques may
be considered to be an "isolated" polypeptide to the extent that it
has been separated from other components a) with which it is
associated in nature; and/or b) with which it was associated when
initially produced.
[0053] The phrase "non-human animal" as used herein refers to a
vertebrate organism that is not a human. In some embodiments, a
non-human animal is a cyclostome, a bony fish, a cartilaginous fish
(e.g., a shark or a ray), an amphibian, a reptile, a mammal, or a
bird. In some embodiments, a non-human mammal is a primate, a goat,
a sheep, a pig, a dog, a cow, or a rodent. In some embodiments, a
non-human animal is a rodent such as a rat or a mouse.
[0054] The phrase "nucleic acid", as used herein, in its broadest
sense, refers to any compound and/or substance that is or can be
incorporated into an oligonucleotide chain. In some embodiments, a
nucleic acid is a compound and/or substance that is or can be
incorporated into an oligonucleotide chain via a phosphodiester
linkage. As will be clear from context, in some embodiments,
"nucleic acid" refers to one or more individual nucleic acid
residues (e.g., nucleotides and/or nucleosides); in some
embodiments, "nucleic acid" refers to an oligonucleotide chain
comprising individual nucleic acid residues. In some embodiments, a
"nucleic acid" is or comprises RNA; in some embodiments, a "nucleic
acid" is or comprises DNA. In some embodiments, a nucleic acid is,
comprises, or consists of one or more natural nucleic acid
residues. In some embodiments, a nucleic acid is, comprises, or
consists of one or more analogs of a natural nucleic acid residue.
In some embodiments, a nucleic acid analog differs from a natural
nucleic acid residue in that it does not utilize a phosphodiester
backbone. For example, in some embodiments, a nucleic acid is,
comprises, or consists of one or more "peptide nucleic acids",
which are known in the art and have peptide bonds instead of
phosphodiester bonds in the backbone, are considered within the
scope of the present invention. Alternatively or additionally, in
some embodiments, a nucleic acid has one or more phosphorothioate
and/or 5'-N-phosphoramidite linkages rather than phosphodiester
bonds. In some embodiments, a nucleic acid is, comprises, or
consists of one or more natural nucleosides (e.g., adenosine,
thymidine, guanosine, cytidine, uridine, deoxyadenosine,
deoxythymidine, deoxyguanosine, and deoxycytidine). In some
embodiments, a nucleic acid is, comprises, or consists of one or
more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine,
inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine,
C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine,
C5-bromouridine, C5-fluorouridine, C5-iodouridine,
C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine,
2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine,
8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine,
methylated bases, intercalated bases, and combinations thereof). In
some embodiments, a nucleic acid comprises one or more modified
sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose,
and hexose) as compared with those in natural nucleic acids (i.e.,
comprises one or more analogs of a natural nucleoside sugar). In
some embodiments, a nucleic acid has a nucleotide sequence that
encodes a functional gene product such as an RNA or protein. In
some embodiments, a nucleic acid has a nucleotide sequence that
includes one or more introns. Those of ordinary skill in the art
will appreciate that a variety of technologies are available and
known in the art for the production of nucleic acids. For example,
in some embodiments, nucleic acids are prepared by a method
selected from the group consisting of isolation from a natural
source, enzymatic synthesis by polymerization based on a
complementary template (in vivo or in vitro), reproduction in a
recombinant cell or system, chemical synthesis, and combinations
thereof. In some embodiments, a nucleic acid is at least 3, 4, 5,
6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,
80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,
20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500,
600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500,
5000 or more residues long. In some embodiments, a nucleic acid is
single stranded; in some embodiments, a nucleic acid is partially
or fully double stranded (i.e., comprises at least two individual
nucleic acid strands whose sequences include complementary elements
that hybridize to one another). In some embodiments a nucleic acid
has a nucleotide sequence comprising at least one element that
encodes, or is the complement of a sequence that encodes, a
polypeptide. In some embodiments, a nucleic acid has enzymatic
activity.
[0055] The phrase "operably linked", as used herein, refers to a
physical juxtaposition (e.g., in three-dimensional space) of
components or elements that interact, directly or indirectly with
one another, or otherwise coordinate with each other to participate
in a biological event, which juxtaposition achieves or permits such
interaction and/or coordination. To give but one example. A control
sequence (e.g., an expression control sequence) in a nucleic acid
is said to be "operably linked" to a coding sequence when it is
located relative to the coding sequence such that its presence or
absence impacts expression and/or activity of the coding sequence.
In many embodiments, "operable linkage" involves covalent linkage
of relevant components or elements with one another. Those skilled
in the art will readily appreciate, however, that in some
embodiments, covalent linkage is not required to achieve effective
operable linkage. For example, in some embodiments, nucleic acid
control sequences that are operably linked with coding sequences
that they control are contiguous with the gene of interest.
Alternatively or additionally, in some embodiments, one or more
such control sequences acts in trans or at a distance to control a
coding sequence of interest. In some embodiments, the term
"expression control sequence" as used herein refers to
polynucleotide sequences which are necessary and/or sufficient to
effect the expression and processing of coding sequences to which
they are ligated. In some embodiments, expression control sequences
may be or comprise appropriate transcription initiation,
termination, promoter and/or enhancer sequences; efficient RNA
processing signals such as splicing and polyadenylation signals;
sequences that stabilize cytoplasmic mRNA; sequences that enhance
translation efficiency (e.g., Kozak consensus sequence); sequences
that enhance protein stability; and/or, in some embodiments,
sequences that enhance protein secretion. In some embodiments, one
or more control sequences is preferentially or exclusively active
in a particular host cell or organism, or type thereof. To give but
one example, in prokaryotes, control sequences typically include
promoter, ribosomal binding site, and transcription termination
sequence; in eukaryotes, in many embodiments, control sequences
typically include promoters, enhancers, and/or transcription
termination sequences. Those of ordinary skill in the art will
appreciate from context that, in many embodiments, the term
"control sequences" refers to components whose presence is
essential for expression and processing, and in some embodiments
includes components whose presence is advantageous for expression
(including, for example, leader sequences, targeting sequences,
and/or fusion partner sequences).
[0056] The term "polypeptide", as used herein, refers to any
polymeric chain of amino acids. In some embodiments, a polypeptide
has an amino acid sequence that occurs in nature. In some
embodiments, a polypeptide has an amino acid sequence that does not
occur in nature. In some embodiments, a polypeptide has an amino
acid sequence that is engineered in that it is designed and/or
produced through action of the hand of man.
[0057] The term "recombinant", as used herein, is intended to refer
to polypeptides (e.g., B cell activating factor proteins as
described herein) that are designed, engineered, prepared,
expressed, created or isolated by recombinant means, such as
polypeptides expressed using a recombinant expression vector
transfected into a host cell, polypeptides isolated from a
recombinant, combinatorial human polypeptide library (Hoogenboom H.
R., (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith W. E.,
(2002) Clin. Biochem. 35:425-445; Gavilondo J. V., and Larrick J.
W. (2002) BioTechniques 29:128-145; Hoogenboom H., and Chames P.
(2000) Immunology Today 21:371-378), antibodies isolated from an
animal (e.g., a mouse) that is transgenic for human immunoglobulin
genes (see e.g., Taylor, L. D., et al. (1992) Nucl. Acids Res.
20:6287-6295; Kellermann S-A., and Green L. L. (2002) Current
Opinion in Biotechnology 13:593-597; Little M. et al (2000)
Immunology Today 21:364-370) or polypeptides prepared, expressed,
created or isolated by any other means that involves splicing
selected sequence elements to one another. In some embodiments, one
or more of such selected sequence elements is found in nature. In
some embodiments, one or more of such selected sequence elements is
designed in silico. In some embodiments, one or more such selected
sequence elements results from mutagenesis (e.g., in vivo or in
vitro) of a known sequence element, e.g., from a natural or
synthetic source. For example, in some embodiments, a recombinant
polypeptide is comprised of sequences found in the genome of a
source organism of interest (e.g., human, mouse, etc.). In some
embodiments, a recombinant polypeptide has an amino acid sequence
that resulted from mutagenesis (e.g., in vitro or in vivo, for
example in a non-human animal), so that the amino acid sequences of
the recombinant polypeptides are sequences that, while originating
from and related to polypeptides sequences, may not naturally exist
within the genome of a non-human animal in vivo.
[0058] The term "replacement" is used herein to refer to a process
through which a "replaced" nucleic acid sequence (e.g., a gene)
found in a host locus (e.g., in a genome) is removed from that
locus and a different, "replacement" nucleic acid is located in its
place. In some embodiments, the replaced nucleic acid sequence and
the replacement nucleic acid sequences are comparable to one
another in that, for example, they are homologous to one another
and/or contain corresponding elements (e.g., protein-coding
elements, regulatory elements, etc.). In some embodiments, a
replaced nucleic acid sequence includes one or more of a promoter,
an enhancer, a splice donor site, a splice receiver site, an
intron, an exon, an untranslated region (UTR); in some embodiments,
a replacement nucleic acid sequence includes one or more coding
sequences. In some embodiments, a replacement nucleic acid sequence
is a homolog of the replaced nucleic acid sequence. In some
embodiments, a replacement nucleic acid sequence is an ortholog of
the replaced sequence. In some embodiments, a replacement nucleic
acid sequence is or comprises a human nucleic acid sequence. In
some embodiments, including where the replacement nucleic acid
sequence is or comprises a human nucleic acid sequence, the
replaced nucleic acid sequence is or comprises a rodent sequence
(e.g., a mouse sequence). The nucleic acid sequence so placed may
include one or more regulatory sequences that are part of source
nucleic acid sequence used to obtain the sequence so placed (e.g.,
promoters, enhancers, 5'- or 3'-untranslated regions, etc.). For
example, in various embodiments, the replacement is a substitution
of an endogenous sequence with a heterologous sequence that results
in the production of a gene product from the nucleic acid sequence
so placed (comprising the heterologous sequence), but not
expression of the endogenous sequence; the replacement is of an
endogenous genomic sequence with a nucleic acid sequence that
encodes a protein that has a similar function as a protein encoded
by the endogenous sequence (e.g., the endogenous genomic sequence
encodes a Baff protein, and the DNA fragment encodes one or more
human BAFF proteins). In various embodiments, an endogenous gene or
fragment thereof is replaced with a corresponding human gene or
fragment thereof. A corresponding human gene or fragment thereof is
a human gene or fragment that is an ortholog of, or is
substantially similar or the same in structure and/or function, as
the endogenous gene or fragment thereof that is replaced.
[0059] The phrase "B-cell activating factor" or "BAFF" or "Baff" as
used herein refers to a tumor necrosis family ligand, e.g., a TNF
family ligand. BAFF is a type II membrane-bound protein, which can
be released as a soluble ligand upon proteolytic processing at a
furin cleavage site. BAFF proteins can form multimers (e.g.,
timers) depending on pH conditions. This characteristic is may be
important for receptor binding. BAFF is expressed on the surface of
a cell and serves as a regulatory protein involved in interactions
between membrane surface proteins on immune cells, e.g., B cells.
Several variants, including those resulting from alternative
splicing events, have been described in human subjects as well as
in rodents. By way of illustration, nucleotide and amino acid
sequences of mouse and human BAFF genes are provided in Table 3.
Persons of skill upon reading this disclosure will recognize that
one or more endogenous Baff genes in a genome (or all) can be
replaced by one or more heterologous Baff genes (e.g., polymorphic
variants, subtypes or mutants, genes from another species,
humanized forms, etc.).
[0060] A "BAFF-expressing cell" as used herein refers to a cell
that expresses a B-cell activating factor ligand. In some
embodiments, a BAFF-expressing cell expresses a B-cell activating
factor ligand on its surface. In some embodiments, a BAFF protein
is expressed on the surface of the cell in an amount sufficient to
mediate cell-to-cell interactions via the BAFF protein expressed on
the surface of the cell. In some embodiments, a BAFF-expressing
cell express a B-cell activating factor ligand in soluble form
(i.e., not on the surface of a cell). Exemplary BAFF-expressing
cells include, but are not limited to, astrocytes, dendritic cells,
monocytes, neutrophils and stromal cells. BAFF interacts with
receptors found predominantly on B cell lineages and is involved in
the activation and survival of B cells. In some embodiments,
non-human animals of the present invention demonstrate immune cell
regulation via humanized Baff ligands expressed on the surface of
one more cells of the non-human animal. In some embodiments,
non-human animals of the present invention promote the long-term
survival of B cells in non-human animals that comprise heterologous
hematopoietic stem cells (e.g., human). In some embodiments,
non-human animals of the present invention promote the long-term
survival of antigen-specific B cells in non-human animals that
comprise heterologous hematopoietic stem cells (e.g., human).
[0061] The term "substantially" as used herein refers to the
qualitative condition of exhibiting total or near-total extent or
degree of a characteristic or property of interest. One of ordinary
skill in the biological arts will understand that biological and
chemical phenomena rarely, if ever, go to completion and/or proceed
to completeness or achieve or avoid an absolute result. The term
"substantially" is therefore used herein to capture the potential
lack of completeness inherent in many biological and chemical
phenomena.
[0062] The phrase "substantial homology" as used herein refers to a
comparison between amino acid or nucleic acid sequences. As will be
appreciated by those of ordinary skill in the art, two sequences
are generally considered to be "substantially homologous" if they
contain homologous residues in corresponding positions. Homologous
residues may be identical residues. Alternatively, homologous
residues may be non-identical residues will appropriately similar
structural and/or functional characteristics. For example, as is
well known by those of ordinary skill in the art, certain amino
acids are typically classified as "hydrophobic" or "hydrophilic"
amino acids, and/or as having "polar" or "non-polar" side chains.
Substitution of one amino acid for another of the same type may
often be considered a "homologous" substitution. Typical amino acid
categorizations are summarized in Table 1 and 2.
[0063] As is well known in this art, amino acid or nucleic acid
sequences may be compared using any of a variety of algorithms,
including those available in commercial computer programs such as
BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and
PSI-BLAST for amino acid sequences. Exemplary such programs are
described in Altschul, et al., Basic local alignment search tool,
J. Mol. Biol., 215(3): 403-410, 1990; Altschul, et al., Methods in
Enzymology; Altschul, et al., "Gapped BLAST and PSI-BLAST: a new
generation of protein database search programs", Nucleic Acids Res.
25:3389-3402, 1997; Baxevanis, et al., Bioinformatics: A Practical
Guide to the Analysis of Genes and Proteins, Wiley, 1998; and
Misener, et al., (eds.), Bioinformatics Methods and Protocols
(Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In
addition to identifying homologous sequences, the programs
mentioned above typically provide an indication of the degree of
homology. In some embodiments, two sequences are considered to be
substantially homologous if at least 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
of their corresponding residues are homologous over a relevant
stretch of residues. In some embodiments, the relevant stretch is a
complete sequence. In some embodiments, the relevant stretch is at
least 9, 10, 11, 12, 13, 14, 15, 16, 17 or more residues. In some
embodiments, the relevant stretch includes contiguous residues
along a complete sequence. In some embodiments, the relevant
stretch includes discontinuous residues along a complete sequence.
In some embodiments, the relevant stretch is at least 10, 15, 20,
25, 30, 35, 40, 45, 50, or more residues.
TABLE-US-00001 TABLE 1 Alanine Ala A nonpolar neutral 1.8 Arginine
Arg R polar positive -4.5 Asparagine Asn N polar neutral -3.5
Aspartic acid Asp D polar negative -3.5 Cysteine Cys C nonpolar
neutral 2.5 Glutamic acid Glu E polar negative -3.5 Glutamine Gln Q
polar neutral -3.5 Glycine Gly G nonpolar neutral -0.4 Histidine
His H polar positive -3.2 Isoleucine Ile I nonpolar neutral 4.5
Leucine Leu L nonpolar neutral 3.8 Lysine Lys K polar positive -3.9
Methionine Met M nonpolar neutral 1.9 Phenylalanine Phe F nonpolar
neutral 2.8 Proline Pro P nonpolar neutral -1.6 Serine Ser S polar
neutral -0.8 Threonine Thr T polar neutral -0.7 Tryptophan Trp W
nonpolar neutral -0.9 Tyrosine Tyr Y polar neutral -1.3 Valine Val
V nonpolar neutral 4.2
TABLE-US-00002 TABLE 2 Ambiguous Amino Acids 3-Letter 1-Letter
Asparagine or aspartic acid Asx B Glutamine or glutamic acid Glx Z
Leucine or Isoleucine Xle J Unspecified or unknown amino acid Xaa
X
[0064] The phrase "substantial identity" as used herein refers to a
comparison between amino acid or nucleic acid sequences. As will be
appreciated by those of ordinary skill in the art, two sequences
are generally considered to be "substantially identical" if they
contain identical residues in corresponding positions. As is well
known in this art, amino acid or nucleic acid sequences may be
compared using any of a variety of algorithms, including those
available in commercial computer programs such as BLASTN for
nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for
amino acid sequences. Exemplary such programs are described in
Altschul, et al., Basic local alignment search tool, J. Mol. Biol.,
215(3): 403-410, 1990; Altschul, et al., Methods in Enzymology;
Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis
et al., Bioinformatics: A Practical Guide to the Analysis of Genes
and Proteins, Wiley, 1998; and Misener, et al., (eds.),
Bioinformatics Methods and Protocols (Methods in Molecular Biology,
Vol. 132), Humana Press, 1999. In addition to identifying identical
sequences, the programs mentioned above typically provide an
indication of the degree of identity. In some embodiments, two
sequences are considered to be substantially identical if at least
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or more of their corresponding residues are
identical over a relevant stretch of residues. In some embodiments,
the relevant stretch is a complete sequence. In some embodiments,
the relevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45,
50, or more residues.
[0065] The phrase "targeting vector" or "targeting construct" as
used herein refers to a polynucleotide molecule that comprises a
targeting region. A targeting region comprises a sequence that is
identical or substantially identical to a sequence in a target
cell, tissue or animal and provides for integration of the
targeting construct into a position within the genome of the cell,
tissue or animal via homologous recombination. Targeting regions
that target using site-specific recombinase recognition sites
(e.g., LoxP or Frt sites) are also included. In some embodiments, a
targeting construct of the present invention further comprises a
nucleic acid sequence or gene of particular interest, a selectable
marker, control and or regulatory sequences, and other nucleic acid
sequences that allow for recombination mediated through exogenous
addition of proteins that aid in or facilitate recombination
involving such sequences. In some embodiments, a targeting
construct of the present invention further comprises a gene of
interest in whole or in part, wherein the gene of interest is a
heterologous gene that encodes a protein in whole or in part that
has a similar function as a protein encoded by an endogenous
sequence.
[0066] The term "variant", as used herein, refers to an entity that
shows significant structural identity with a reference entity but
differs structurally from the reference entity in the presence or
level of one or more chemical moieties as compared with the
reference entity. In many embodiments, a variant also differs
functionally from its reference entity. In general, whether a
particular entity is properly considered to be a "variant" of a
reference entity is based on its degree of structural identity with
the reference entity. As will be appreciated by those skilled in
the art, any biological or chemical reference entity has certain
characteristic structural elements. A variant, by definition, is a
distinct chemical entity that shares one or more such
characteristic structural elements. To give but a few examples, a
small molecule may have a characteristic core structural element
(e.g., a macrocycle core) and/or one or more characteristic pendent
moieties so that a variant of the small molecule is one that shares
the core structural element and the characteristic pendent moieties
but differs in other pendent moieties and/or in types of bonds
present (single vs. double, E vs. Z, etc.) within the core, a
polypeptide may have a characteristic sequence element comprised of
a plurality of amino acids having designated positions relative to
one another in linear or three-dimensional space and/or
contributing to a particular biological function, a nucleic acid
may have a characteristic sequence element comprised of a plurality
of nucleotide residues having designated positions relative to on
another in linear or three-dimensional space. For example, a
variant polypeptide may differ from a reference polypeptide as a
result of one or more differences in amino acid sequence and/or one
or more differences in chemical moieties (e.g., carbohydrates,
lipids, etc.) covalently attached to the polypeptide backbone. In
some embodiments, a variant polypeptide shows an overall sequence
identity with a reference polypeptide that is at least 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%.
Alternatively or additionally, in some embodiments, a variant
polypeptide does not share at least one characteristic sequence
element with a reference polypeptide. In some embodiments, the
reference polypeptide has one or more biological activities. In
some embodiments, a variant polypeptide shares one or more of the
biological activities of the reference polypeptide. In some
embodiments, a variant polypeptide lacks one or more of the
biological activities of the reference polypeptide. In some
embodiments, a variant polypeptide shows a reduced level of one or
more biological activities as compared with the reference
polypeptide. In many embodiments, a polypeptide of interest is
considered to be a "variant" of a parent or reference polypeptide
if the polypeptide of interest has an amino acid sequence that is
identical to that of the parent but for a small number of sequence
alterations at particular positions. Typically, fewer than 20%,
15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% of the residues in the
variant are substituted as compared with the parent. In some
embodiments, a variant has 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1
substituted residue as compared with a parent. Often, a variant has
a very small number (e.g., fewer than 5, 4, 3, 2, or 1) number of
substituted functional residues (i.e., residues that participate in
a particular biological activity). Furthermore, a variant typically
has not more than 5, 4, 3, 2, or 1 additions or deletions, and
often has no additions or deletions, as compared with the parent.
Moreover, any additions or deletions are typically fewer than about
25, about 20, about 19, about 18, about 17, about 16, about 15,
about 14, about 13, about 10, about 9, about 8, about 7, about 6,
and commonly are fewer than about 5, about 4, about 3, or about 2
residues. In some embodiments, the parent or reference polypeptide
is one found in nature. As will be understood by those of ordinary
skill in the art, a plurality of variants of a particular
polypeptide of interest may commonly be found in nature,
particularly when the polypeptide of interest is an infectious
agent polypeptide.
[0067] The term "vector", as used herein, refers to a nucleic acid
molecule capable of transporting another nucleic acid to which it
is associated. In some embodiment, vectors are capable of
extra-chromosomal replication and/or expression of nucleic acids to
which they are linked in a host cell such as a eukaryotic and/or
prokaryotic cell. Vectors capable of directing the expression of
operatively linked genes are referred to herein as "expression
vectors."
[0068] The term "wild-type", as used herein, has its art-understood
meaning that refers to an entity having a structure and/or activity
as found in nature in a "normal" (as contrasted with mutant,
diseased, altered, etc.) state or context. Those of ordinary skill
in the art will appreciate that wild type genes and polypeptides
often exist in multiple different forms (e.g., alleles).
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
[0069] The present invention provides, among other things, improved
and/or engineered non-human animals having humanized genetic
material encoding a B-cell activating factor protein (e.g., Baff).
In certain embodiments, such non-human animals are useful, for
example, for assays in transplant engraftment, B cell activation
and survival of antigen-specific B cells post immunization. It is
contemplated that such non-human animals provide an improvement in
B cell activation and survival of antigen-specific B cells post
immunization post-engraftment of human hematopoietic stem cells.
Therefore, the present invention is particularly useful for
maintaining human hematopoietic cells in non-human animals. In
particular, the present invention encompasses the humanization of a
rodent Baff gene resulting in expression of a humanized protein on
the plasma membrane surface of cells of the non-human animal. Such
humanized proteins have the capacity to recognize engrafted human
cells via engagement of humanized Baff proteins and
ligands/receptors present on the surface of the engrafted human
cells. In some embodiments, non-human animals of the present
invention are capable of receiving transplanted human hematopoietic
cells; in some embodiments, such non-human mammals develop and/or
have an immune system comprising human cells. In some embodiments,
humanized Baff proteins have sequence encoded by exons 3 to 6 of a
human BAFF gene. In some embodiments, non-human animals of the
present invention comprise a genetically modified Baff gene that
contains genetic material from the non-human animal and a
heterologous species (e.g., a human). In some embodiments,
non-human animals of the present invention comprise a humanized
Baff gene, wherein the humanized Baff gene comprises exons 3, 4, 5
and 6 of a human BAFF gene. In some embodiments, the expression of
the humanized Baff protein is under the control of non-human Baff
genetic material (e.g., a non-human Baff promoter).
[0070] Various aspects of the invention are described in detail in
the following sections. The use of sections is not meant to limit
the invention. Each section can apply to any aspect of the
invention. In this application, the use of "or" means "and/or"
unless stated otherwise.
[0071] B-Cell Activating Factor (BAFF) Gene
[0072] B-cell activating factor (BAFF or Baff) is a member of the
tumor necrosis factor (TNF) ligand superfamily and is expressed by
many different cell types including, but not limited to,
astrocytes, B cell lineage cells, dendritic cells, monocytes,
neutrophils and stromal cells. BAFF (also referred to as tumor
necrosis factor ligand superfamily member 13C, TNFSF13C, BAFF,
BLYS, CD257, DTL, TALL-1, TALL1, THANK, TNFSF20 and ZTNF4) is
expressed on the cell surface as a Type II transmembrane protein
and can be released in soluble form via cleavage at a furin
consensus site after proteolysis. Soluble BAFF can exist in
multiple forms (e.g., timers, 60-mers) depending upon pH. The gene
structure for BAFF in mouse and man differ slightly in that the
former contains an additional exon. In humans, exon 1 encodes the
transmembrane domain, exon 2 encodes the furin cleavage site, and
exons 3 to 6 encode the TNF domain, which is responsible for
receptor binding. In mouse, exon 1 encodes the transmembrane
domain, exon 2 encodes the furin cleavage site, exon 3 encodes
additional amino acids between the furin site and the TNF domain,
and exons 4-7 encode the TNF domain. For both mouse and man,
alternative splice variants result in a deletion of an interior
portion of the protein which yield a variant referred to as
"delta-BAFF" (or .DELTA.BAFF). In humans, it is exon 3 that is
skipped, whereas in mouse it is exon 4 that is skipped. .DELTA.BAFF
is still expressed on the cell surface, however, release of the
soluble form is reportedly prevented. Reported receptors for BAFF
include, most notably, BAFF receptor (BAFF-R), but also include
transmembrane activator and calcium modulator and cyclophilin
ligand interactor (TACI) and B cell maturation antigen (BCMA). BAFF
binds to both BAFF-R and TACI with strong affinity, whereas BAFF
binds to BCMA with weak affinity.
[0073] The role of Baff, in particular, has been investigated in
respect of its role in the activation and differentiation of B
cells. For example, elevated levels of Baff in transgenic mice
overexpressing mouse Baff were found to promote the survival,
tolerance and rescue of B cells with affinity for self-antigens
thereby promoting autoantibody secretion (Ota et al., 2010, J.
Immunol. 185:4128-4136).
[0074] BAFF Sequences
[0075] Exemplary BAFF sequences for human and mouse are set forth
in Table 3. For cDNA sequences, consecutive exons are separated by
alternating underlined text. For protein sequences, predicted
transmembrane regions are underlined.
TABLE-US-00003 TABLE 3 Mouse Baff cDNA
GGCACGAGGCAGATTGAGCAATCCATGGAAGGCCAGAGCCAGAGAACCTA NM_033622.1
CTTCAGGGTAGCAAAAGATGCAGAAGAAAGTCAGGAGAGCGCTCCTGGGG
GAACCCAGCCCTGCCATGCTCTGAGGGCAGTCTCCCAGGACACAGATGAC
AGGAAATGACCCACCCCTGTGGTCACTTACTCCAAAGGCCTAGACCTTCA
AAGTGCTCCTCGTGGAATGGATGAGTCTGCAAAGACCCTGCCACCACCGT
GCCTCTGTTTTTGCTCCGAGAAAGGAGAAGATATGAAAGTGGGATATGAT
CCCATCACTCCGCAGAAGGAGGAGGGTGCCTGGTTTGGGATCTGCAGGGA
TGGAAGGCTGCTGGCTGCTACCCTCCTGCTGGCCCTGTTGTCCAGCAGTT
TCACAGCGATGTCCTTGTACCAGTTGGCTGCCTTGCAAGCAGACCTGATG
AACCTGCGCATGGAGCTGCAGAGCTACCGAGGTTCAGCAACACCAGCCGC
CGCGGGTGCTCCAGAGTTGACCGCTGGAGTCAAACTCCTGACACCGGCAG
CTCCTCGACCCCACAACTCCAGCCGCGGCCACAGGAACAGACGCGCTTTC
CAGGGACCAGAGGAAACAGAACAAGATGTAGACCTCTCAGCTCCTCCTGC
ACCATGCCTGCCTGGATGCCGCCATTCTCAACATGATGATAATGGAATGA
ACCTCAGAAACATCATTCAAGACTGTCTGCAGCTGATTGCAGACAGCGAC
ACGCCGACTATACGAAAAGGAACTTACACATTTGTTCCATGGCTTCTCAG
CTTTAAAAGAGGAAATGCCTTGGAGGAGAAAGAGAACAAAATAGTGGTGA
GGCAAACAGGCTATTTCTTCATCTACAGCCAGGTTCTATACACGGACCCC
ATCTTTGCTATGGGTCATGTCATCCAGAGGAAGAAAGTACACGTCTTTGG
GGACGAGCTGAGCCTGGTGACCCTGTTCCGATGTATTCAGAATATGCCCA
AAACACTGCCCAACAATTCCTGCTACTCGGCTGGCATCGCGAGGCTGGAA
GAAGGAGATGAGATTCAGCTTGCAATTCCTCGGGAGAATGCACAGATTTC
ACGCAACGGAGACGACACCTTCTTTGGTGCCCTAAAACTGCTGTAACTCA
CTTGCTGGAGTGCGTGATCCCCTTCCCTCGTCTTCTCTGTACCTCCGAGG
GAGAAACAGACGACTGGAAAAACTAAAAGATGGGGAAAGCCGTCAGCGAA
AGTTTTCTCGTGACCCGTTGAATCTGATCCAAACCAGGAAATATAACAGA
CAGCCACAACCGAAGTGTGCCATGTGAGTTATGAGAAACGGAGCCCGCGC
TCAGAAAGACCGGATGAGGAAGACCGTTTTCTCCAGTCCTTTGCCAACAC
GCACCGCAACCTTGCTTTTTGCCTTGGGTGACACATGTTCAGAATGCAGG
GAGATTTCCTTGTTTTGCGATTTGCCATGAGAAGAGGGCCCACAACTGCA
GGTCACTGAAGCATTCACGCTAAGTCTCAGGATTTACTCTCCCTTCTCAT
GCTAAGTACACACACGCTCTTTTCCAGGTAATACTATGGGATACTATGGA
AAGGTTGTTTGTTTTTAAATCTAGAAGTCTTGAACTGGCAATAGACAAAA
ATCCTTATAAATTCAAGTGTAAAATAAACTTAATTAAAAAGGTTTAAGTG TGAAAAAAAA (SEQ
ID NO: 1) Mouse Baff Protein
MDESAKTLPPPCLCFCSEKGEDMKVGYDPITPQKEEGAWFGICRDGRLLA NP_296371.1
ATLLLALLSSSFTAMSLYQLAALQADLMNLRMELQSYRGSATPAAAGAPE
LTAGVKLLTPAAPRPHNSSRGHRNRRAFQGPEETEQDVDLSAPPAPCLPG
CRHSQHDDNGMNLRNIIQDCLQLIADSDTPTIRKGTYTFVPWLLSFKRGN
ALEEKENKIVVRQTGYFFIYSQVLYTDPIFAMGHVIQRKKVHVFGDELSL
VTLFRCIQNMPKTLPNNSCYSAGIARLEEGDEIQLAIPRENAQISRNGDD TFFGALKLL (SEQ
ID NO: 2) Mouse .DELTA.Baff Protein
MDESAKTLPPPCLCFCSEKGEDMKVGYDPITPQKEEGAWFGICRDGRLLA AY290823.1
ATLLLALLSSSFTAMSLYQLAALQADLMNLRMELQSYRGSATPAAAGAPE
LTAGVKLLTPAAPRPHNSSRGHRNRRAFQGPEETEQDVDLSAPPAPCLPG
CRHSQHDDNGMNLRNRTYTFVPWLLSFKRGNALEEKENKIVVRQTGYFFI
YSQVLYTDPIFAMGHVIQRKKVHVFGDELSLVTLFRCIQNMPKTLPNNSC
YSAGIARLEEGDEIQLAIPRENAQISRNGDDTFFGALKLL (SEQ ID NO: 3) Human BAFF
cDNA GAAATTCTTACAAAAACTGAAAGTGAAATGAGGAAGACAGATTGAGCAAT NM_006573.4
CCAATCGGAGGGTAAATGCCAGCAAACCTACTGTACAGTAGGGGTAGAGA
TGCAGAAAGGCAGAAAGGAGAAAATTCAGGATAACTCTCCTGAGGGGTGA
GCCAAGCCCTGCCATGTAGTGCACGCAGGACATCAACAAACACAGATAAC
AGGAAATGATCCATTCCCTGTGGTCACTTATTCTAAAGGCCCCAACCTTC
AAAGTTCAAGTAGTGATATGGATGACTCCACAGAAAGGGAGCAGTCACGC
CTTACTTCTTGCCTTAAGAAAAGAGAAGAAATGAAACTGAAGGAGTGTGT
TTCCATCCTCCCACGGAAGGAAAGCCCCTCTGTCCGATCCTCCAAAGACG
GAAAGCTGCTGGCTGCAACCTTGCTGCTGGCACTGCTGTCTTGCTGCCTC
ACGGTGGTGTCTTTCTACCAGGTGGCCGCCCTGCAAGGGGACCTGGCCAG
CCTCCGGGCAGAGCTGCAGGGCCACCACGCGGAGAAGCTGCCAGCAGGAG
CAGGAGCCCCCAAGGCCGGCCTGGAGGAAGCTCCAGCTGTCACCGCGGGA
CTGAAAATCTTTGAACCACCAGCTCCAGGAGAAGGCAACTCCAGTCAGAA
CAGCAGAAATAAGCGTGCCGTTCAGGGTCCAGAAGAAACAGTCACTCAAG
ACTGCTTGCAACTGATTGCAGACAGTGAAACACCAACTATACAAAAAGGA
TCTTACACATTTGTTCCATGGCTTCTCAGCTTTAAAAGGGGAAGTGCCCT
AGAAGAAAAAGAGAATAAAATATTGGTCAAAGAAACTGGTTACTTTTTTA
TATATGGTCAGGTTTTATATACTGATAAGACCTACGCCATGGGACATCTA
ATTCAGAGGAAGAAGGTCCATGTCTTTGGGGATGAATTGAGTCTGGTGAC
TTTGTTTCGATGTATTCAAAATATGCCTGAAACACTACCCAATAATTCCT
GCTATTCAGCTGGCATTGCAAAACTGGAAGAAGGAGATGAACTCCAACTT
GCAATACCAAGAGAAAATGCACAAATATCACTGGATGGAGATGTCACATT
TTTTGGTGCATTGAAACTGCTGTGACCTACTTACACCATGTCTGTAGCTA
TTTTCCTCCCTTTCTCTGTACCTCTAAGAAGAAAGAATCTAACTGAAAAT
ACCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGTAGTTACCATTGCCT
TTTCTGTGAGCTATTTGTTTTGGTTTGCTGAAACTAGTCCAAAACAGGAA
ATTTAACAGACAGCCACAGCCAAAGAGTGTCATGTGAATTACAAGAAATA
GAGCCCATTTAGGGAAAGATAGAACTAGAAAGGCTTTTCATTATAATTCC
ATGTTGAACAATTGAGTCATAGCTTCTTATCTTGGAGGAAGGACACAATT
CAAAGGGGCAGTAAGGATTTTGTAAAACGTGGCATCCATAATTTACTATG
GAGCAAGTGCCCACATCTCTAGGACATTAAGACATTTATGAGAAATCTCA
GGATTCATCTTCTGTTTTTATGTTAAATGCACTCCCTCCTTTTCAGTTAA
CATTATAAAAAGTAAAAAATGAAAATTTTAGAAATCTTGCATTAGACACA
TGAAAAAATAACTAAAAGTTTAAATTTAAATATGAAACAATTTTGCTGAA
AATAGTATCCATATACTATTTAAGTCTTTTATGGTTATTTCAAGTATACA
ATTTCTATCTGTAATGTAATATATTACCCACACATTTTTTTCACAGGAGA
GAGAGAATATCCTCATTTGTTTATGCTCATGTGTATTTTCTATAGTGAAT
TTCAGAAACTTTTAATATCAGGTAATTTCAATTTATGCCTATAAAGCATT
GATTGAAAAATAACTAGAATTGTGCATATATAACACATAATCTCCAACAG
AAGTTACTGAATACATTCATACTAATGTAATGTAATTTCCCTTTATTTCT
TGCTCTTCTGTTTCAAACTGCTGCTATTGTAGTTTACATATCCCAACCTT
TAAAAATATTCCTCTTATTAGCTTTATATTCACTTTATAGAAGTTGAGTT
TTAATTAAAATTCTTGGCATCCTGAAGTATGTCACATAGCATGTGCTCCT
TATAAATATGTTGATATCTCAGAAGACAGCATCCCGGTTTTCATTTTATA
AAGTACCATACTTAAGAATGCTGTAATACTTATCTTTTATAACATGTTTC
CTTCGCTTTGCTTGTCTTTTATGTCATCAGTTTTAACTGTTTACTTCATT
TAACAGTTTACATCATTCAACAGTTTACTTCATTAAACAGTAGGTGGAAA
AATAGATGCCAGTCTATGAAAATCTTCCCATCTATATCAAAATACTTTTC
AAGGATATACTTTTCAAAACAAACGATTTAAATTTTATGTTTAAAATATA
AACTTTAGATTTAAACTTTATTTAAATATCTGGTTCCTATGATTTTGACT
TCAGTAAGTTCAAATAAAATATATTTTGCAATTCATTTTTACATTATAAT
TTAAAAAGAAGAAGCGATAAGTGGAGTCAGTTTCAATGCTAGGTGGGGTG
GTTAATGATTTTTCTGGTGTTGCTGCTAATGTGGATTAACAAATAAAAAC
ATTCATTGCCTTTTGCCTCATAAAA (SEQ ID NO: 4) Human BAFF Protein
MDDSTEREQSRLTSCLKKREEMKLKECVSILPRKESPSVRSSKDGKLLAA NP_006564.1
TLLLALLSCCLTVVSFYQVAALQGDLASLRAELQGHHAEKLPAGAGAPKA
GLEEAPAVTAGLKIFEPPAPGEGNSSQNSRNKRAVQGPEETVTQDCLQLI
ADSETPTIQKGSYTFVPWLLSFKRGSALEEKENKILVKETGYFFIYGQVL
YTDKTYAMGHLIQRKKVHVFGDELSLVTLFRCIQNMPETLPNNSCYSAGI
AKLEEGDELQLAIPRENAQISLDGDVTFFGALKLL (SEQ ID NO: 5) Human
.DELTA.BAFF Protein
MDDSTEREQSRLTSCLKKREEMKLKECVSILPRKESPSVRSSKDGKLLAA AY302751.1
TLLLALLSCCLTVVSFYQVAALQGDLASLRAELQGHHAEKLPAGAGAPKA
GLEEAPAVTAGLKIFEPPAPGEGNSSQNSRNKRAVQGPEETGSYTFVPWL
LSFKRGSALEEKENKILVKETGYFFIYGQVLYTDKTYAMGHLIQRKKVHV
FGDELSLVTLFRCIQNMPETLPNNSCYSAGIAKLEEGDELQLAIPRENAQ ISLDGDVTFFGALKLL
(SEQ ID NO: 6) Humanized Baff Protein
MDESAKTLPPPCLCFCSEKGEDMKVGYDPITPQKEEGAWFGICRDGRLLA
ATLLLALLSSSFTAMSLYQLAALQADLMNLRMELQSYRGSATPAAAGAPE
LTAGVKLLTPAAPRPHNSSRGHRNRRAFQGPEETVTQDCLQLIADSETPT
IQKGSYTFVPWLLSFKRGSALEEKENKILVKETGYFFIYGQVLYTDKTYA
MGHLIQRKKVHVFGDELSLVTLFRCIQNMPETLPNNSCYSAGIAKLEEGD
ELQLAIPRENAQISLDGDVTFFGALKLL (SEQ ID NO: 7)
[0076] Humanized Baff Non-Human Animals
[0077] Non-human animals are provided that express genetically
modified (e.g., humanized) Baff proteins on the surface of cells
(e.g., dendritic cells) of the non-human animals. Specifically, the
present invention provides non-human animals that express
genetically modified (e.g., humanized) Baff proteins on the surface
of their cells, the proteins being encoded by and/or expressed from
a genetic modification of an endogenous locus of the non-human
animal that encodes a Baff protein. Suitable examples presented
herein specifically exemplify rodents, in particular, mice.
[0078] A genetically modified Baff gene, in some embodiments,
comprises genetic material from a heterologous species (e.g.,
humans), wherein the genetically modified Baff gene encodes a Baff
protein that comprises the encoded portion of the genetic material
from the heterologous species. In some embodiments, a genetically
modified Baff gene of the present invention comprises genomic DNA
of a heterologous species that corresponds to the extracellular
portion of a Baff protein that is expressed on the plasma membrane
of a cell. Non-human animals, embryos, cells and targeting
constructs for making non-human animals, non-human embryos, and
cells containing said genetically modified Baff gene are also
provided.
[0079] In some embodiments, the endogenous Baff gene is deleted. In
some embodiments, the endogenous Baff gene is altered, wherein a
portion of the endogenous Baff gene is replaced with a heterologous
sequence (e.g., a human BAFF gene sequence, in whole or in part).
In some embodiments, all or substantially all of the endogenous
Baff gene is replaced with a heterologous gene (e.g., a human BAFF
gene). In some embodiments, a portion of a heterologous Baff gene
is inserted into an endogenous non-human Baff gene. In some
embodiments, the heterologous gene is a human gene.
[0080] A non-human animal of the present invention contains a human
BAFF gene, in whole or in part, at an endogenous non-human Baff
locus. Thus, such non-human animals can be described as having a
humanized Baff gene. The replaced, inserted or modified endogenous
Baff gene (i.e., the humanized Baff gene) can be detected using a
variety of methods including, for example, PCR, Western blot,
Southern blot, restriction fragment length polymorphism (RFLP), or
a gain or loss of allele assay.
[0081] In various embodiments, a humanized Baff gene according to
the present invention includes a Baff gene that has a third,
fourth, fifth, and sixth exon each having a sequence at least 50%
(e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or more) identical to a third, fourth,
fifth, and sixth exon that appear in a human BAFF gene of Table
3.
[0082] In various embodiments, a humanized Baff gene according to
the present invention includes a Baff gene that has a nucleotide
coding sequence (e.g., a cDNA sequence) at least 50% (e.g., 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more) identical to nucleotides 692-2671 that
appear in a human BAFF cDNA sequence of Table 3.
[0083] In various embodiments, a humanized Baff protein produced by
a non-human animal of the present invention has an extracellular
portion having a sequence that is at least 50% (e.g., 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or more) identical to an extracellular portion of a
human BAFF protein that appears in Table 3.
[0084] In various embodiments, a humanized Baff protein produced by
a non-human animal of the present invention has an extracellular
portion having a sequence that is at least 50% (e.g., 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or more) identical to amino acid residues 142 to 285
that appear in a human BAFF protein of Table 3.
[0085] In various embodiments, a humanized Baff protein produced by
a non-human animal of the present invention has an amino acid
sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more)
identical to an amino acid sequence of a humanized Baff protein
that appears in Table 3.
[0086] In various embodiments, a humanized Baff protein produced by
a non-human animal of the present invention has an amino acid
sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more)
identical to an amino acid sequence of a human BAFF protein that
appears in Table 3.
[0087] Compositions and methods for making non-human animals that
expresses a humanized Baff protein, including specific polymorphic
forms or allelic variants (e.g., single amino acid differences,
alternative splice variants, etc.), are provided, including
compositions and methods for making non-human animals that
expresses such proteins from a human promoter and a human
regulatory sequence or, optionally, from a non-human promoter and a
non-human regulatory sequence. In some embodiments, compositions
and methods for making non-human animals that expresses such
proteins from an endogenous promoter and an endogenous regulatory
sequence are also provided. The methods include inserting the
genetic material encoding a human BAFF protein, in whole or in
part, at a precise location in the genome of a non-human animal
that corresponds to an endogenous Baff gene thereby creating a
humanized Baff gene that expresses a BAFF protein that is human, in
whole or in part. In some embodiments, the methods include
inserting genomic DNA corresponding to exons 3 to 6 of a human BAFF
gene into an endogenous Baff gene of the non-human animal thereby
creating a humanized gene that encodes a Baff protein that contains
a human portion containing amino acids encoded by the inserted
exons.
[0088] A humanized Baff gene approach employs a relatively minimal
modification of the endogenous gene and results in natural
Baff-mediated signal transduction in the non-human animal, in
various embodiments, because the genomic sequence of the Baff gene
is modified in a single fragment and therefore retain normal
functionality by including necessary regulatory sequences. Thus, in
such embodiments, the Baff gene modification does not affect other
surrounding genes or other endogenous Baff genes. Further, in
various embodiments, the modification does not affect the assembly
of a functional transmembrane protein on the plasma membrane and
maintains normal association with its receptors via binding and
interaction of the extracellular portion with a given receptor
which is unaffected by the modification.
[0089] A schematic illustration (not to scale) of endogenous murine
and human BAFF genes is provided in FIG. 1. A schematic
illustration (not to scale) of a humanized Baff gene is provided in
FIG. 2. As illustrated, genomic DNA containing exons 3 to 6 of a
human BAFF gene is inserted into an endogenous murine Baff gene by
a targeting construct. This genomic DNA comprises the portion of
the gene that encodes the extracellular portion (e.g., amino acid
residues 142 to 285) of a human BAFF protein responsible for
receptor binding.
[0090] A non-human animal (e.g., a mouse) having a humanized Baff
gene can be made by any method known in the art. For example, a
targeting vector can be made that introduces a human BAFF gene, in
whole or in part, with a selectable marker gene. FIG. 2 illustrates
a mouse genome comprising an insertion of exons 3 to 6 of a human
BAFF gene. As illustrated, the targeting construct contains unique
5' and 3' restriction endonuclease sites which allow for the
precise insertion of the human genetic material comprising exons 3
to 6 of a human BAFF gene. The targeting construct also contains a
self-deleting drug selection cassette (e.g., a neomycin resistance
gene flanked on both sides by LoxP sequences; see U.S. Pat. No.
8,354,389 and U.S. Pat. No. 8,518,392, both of which are herein
incorporated by reference), which is positioned 3' genetic material
comprising exons 3 to 6 of a human BAFF gene. Upon digestion and
religation, exons 3 to 6 of a human BAFF gene are inserted into an
endogenous murine Baff gene that has been specifically engineered
to accept the human sequence contained in the targeting vector. A
humanized Baff gene is created resulting in a cell or non-human
animal that expresses a humanized Baff protein that contains amino
acids encoded by exons 3 to 6 of a human BAFF gene. The drug
selection cassette will be removed in a development-dependent
manner, i.e., progeny derived from mice whose germ line cells
containing the humanized Baff gene described above will shed the
selectable marker from differentiated cells during development.
[0091] The non-human animals of the present invention may be
prepared as described above, or using methods known in the art, to
comprise additional human or humanized genes, oftentimes depending
on the intended use of the non-human animal. Genetic material of
such additional human or humanized genes may be introduced through
the further alteration of the genome of cells (e.g., embryonic stem
cells) having the genetic modifications as described above or
through breeding techniques known in the art with other genetically
modified strains as desired. In some embodiments, non-human animals
of the present invention are prepared to further comprise one or
more human or humanized genes selected from BAFF-R, TACI, and BCMA.
In some embodiments, non-human animals of the present invention are
prepared to further comprise a human or humanized A
PRoliferation-Inducing Ligand (APRIL) gene. In some embodiments,
non-human animals of the present invention are prepared to further
comprise a human or humanized TNF-related weak inducer of apoptosis
(TWEAK). In some embodiments, non-human animals of the present
invention comprise a humanized Baff gene as described herein and
genetic material from a heterologous species (e.g., humans),
wherein the genetic material encodes, in whole or in part, one or
more heterologous proteins selected from BAFF-R, TACI, BCMA, APRIL
and TWEAK.
[0092] In addition to mice having humanized Baff genes as described
herein, also provided herein are other genetically modified
non-human animals that comprise humanized Baff genes. In some
embodiments, such non-human animals comprise a humanized Baff gene
operably linked to an endogenous Baff promoter sequence. In some
embodiments, such non-human animals express a humanized BAFF
protein from an endogenous Baff locus, wherein the humanized Baff
protein comprises amino acid residues 142 to 285 of a human BAFF
protein.
[0093] Such non-human animals may be selected from the group
consisting of a mouse, rat, rabbit, pig, bovine (e.g., cow, bull,
buffalo), deer, sheep, goat, chicken, cat, dog, ferret, primate
(e.g., marmoset, rhesus monkey). For the non-human animals where
suitable genetically modifiable ES cells are not readily available,
other methods are employed to make a non-human animal comprising
the genetic modifications as described herein. Such methods
include, e.g., modifying a non-ES cell genome (e.g., a fibroblast
or an induced pluripotent cell) and employing nuclear transfer to
transfer the modified genome to a suitable cell, e.g., an oocyte,
and gestating the modified cell (e.g., the modified oocyte) in a
non-human animal under suitable conditions to form an embryo.
[0094] In some embodiments, a non-human animal of the present
invention is a mammal. In some embodiments, a non-human animal of
the present invention is a small mammal, e.g., of the superfamily
Dipodoidea or Muroidea. In some embodiments, a genetically modified
animal of the present invention is a rodent. In some embodiments, a
rodent of the present invention is selected from a mouse, a rat,
and a hamster. In some embodiments, a rodent of the present
invention is selected from the superfamily Muroidea. In some
embodiments, a genetically modified animal of the present invention
is from a family selected from Calomyscidae (e.g., mouse-like
hamsters), Cricetidae (e.g., hamster, New World rats and mice,
voles), Muridae (true mice and rats, gerbils, spiny mice, crested
rats), Nesomyidae (climbing mice, rock mice, with-tailed rats,
Malagasy rats and mice), Platacanthomyidae (e.g., spiny dormice),
and Spalacidae (e.g., mole rates, bamboo rats, and zokors). In some
certain embodiments, a genetically modified rodent of the present
invention is selected from a true mouse or rat (family Muridae), a
gerbil, a spiny mouse, and a crested rat. In some certain
embodiments, a genetically modified mouse of the present invention
is from a member of the family Muridae. In some embodiment, an
non-human animal of the present invention is a rodent. In some
certain embodiments, a rodent of the present invention is selected
from a mouse and a rat. In some embodiments, a non-human animal of
the present invention is a mouse.
[0095] In some embodiments, a non-human animal of the present
invention is a rodent that is a mouse of a C57BL strain selected
from C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J,
C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and
C57BL/O1a. In some certain embodiments, a mouse of the present
invention is a 129 strain selected from the group consisting of a
strain that is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV,
129S1/SvIm), 129S2, 129S4, 129S5, 129S9/SvEvH, 129/SvJae, 129S6
(129/SvEvTac), 129S7, 129S8, 129T1, 129T2 (see, e.g., Festing et
al., 1999, Mammalian Genome 10:836; Auerbach et al., 2000,
Biotechniques 29(5):1024-1028, 1030, 1032). In some certain
embodiments, a genetically modified mouse of the present invention
is a mix of an aforementioned 129 strain and an aforementioned
C57BL/6 strain. In some certain embodiments, a mouse of the present
invention is a mix of aforementioned 129 strains, or a mix of
aforementioned BL/6 strains. In some certain embodiments, a 129
strain of the mix as described herein is a 129S6 (129/SvEvTac)
strain. In some embodiments, a mouse of the present invention is a
BALB strain, e.g., BALB/c strain. In some embodiments, a mouse of
the present invention is a mix of a BALB strain and another
aforementioned strain.
[0096] In some embodiments, a non-human animal of the present
invention is a rat. In some certain embodiments, a rat of the
present invention is selected from a Wistar rat, an LEA strain, a
Sprague Dawley strain, a Fischer strain, F344, F6, and Dark Agouti.
In some certain embodiments, a rat strain as described herein is a
mix of two or more strains selected from the group consisting of
Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark
Agouti.
[0097] Methods Employing Non-Human Animals Having Humanized BAFF
Genes
[0098] Baff transgenic non-human animals (e.g., mice) have been
reported (Mackay et al., 1999, J. Exp. Med. 190(11):1697-1710;
Khare et al., 2000, PNAS 97(7):3370-3375; Gavin et al., 2005, J.
Immunol. 175:319-328). Such animals have been employed in a variety
of assays to determine the molecular aspects of BAFF expression,
function and regulation. However, they are not without limitation.
For example, use of Baff transgenic mice have been limited due to
overexpression of murine Baff (e.g., full-length Baff or
.DELTA.Baff). Overexpression of Baff in transgenic mice leads to
several B cell abnormalities characterized by, inter alia,
excessive accumulation and activation of B cells, and autoimmune
disease through proliferation of auto-reactive B cells. In some
cases, transgenic mice overexpressing murine BAFF have increased
levels of serum immunoglobulin (e.g., IgM, IgG, IgE, etc.).
Further, transgenic mice overexpressing murine Baff demonstrate
other abnormalities such as glomerulonephritis. Therefore, the
molecular aspects of BAFF-mediated biological function and
signaling pathways has not been exploited in transgenic mice.
[0099] Non-human animals of the present invention provide an
improved in vivo system and source of biological materials (e.g.,
cells) expressing human BAFF that are useful for a variety of
assays. In various embodiments, non-human animals of the present
invention are used to develop therapeutics that target human BAFF
and/or modulate BAFF-mediated signaling pathways. In various
embodiments, mice of the present invention are used to screen and
develop candidate therapeutics (e.g., antibodies) that bind to
human BAFF. In various embodiments, non-human animals of the
present invention are used to determine the binding profile of
antagonists and/or agonists a humanized Baff on the surface of a
cell of a non-human animal as described herein.
[0100] In various embodiments, non-human animals of the present
invention are used to measure the therapeutic effect of blocking or
modulating human BAFF signal transduction (e.g., phosphorylation)
and the effect on gene expression as a result of cellular changes.
In various embodiments, non-human animals of the present invention
are used to measure the therapeutic effect of blocking or
modulating human BAFF-BAFFR, BAFF-TACI, and/or BAFF-BCMA signaling
pathways, for example, the modulation of NF-.kappa.B-mediated
transcription of DNA. In various embodiments, a non-human animal of
the present invention or cells isolated therefrom are exposed to a
candidate therapeutic that binds to a human BAFF protein on the
surface of a cell of the non-human animal and, after a subsequent
period of time, analyzed for effects on BAFF-dependent processes,
for example, B activation, regulation of the numbers of specific B
cell subsets in various compartments (e.g., spleen, bone marrow,
lymph node, etc.), survival of auto-reactive B cells, and
NF-.kappa.B activation.
[0101] Non-human animals of the present invention express humanized
Baff protein, thus cells, cell lines, and cell cultures can be
generated to serve as a source of humanized Baff for use in binding
and functional assays, e.g., to assay for binding or function of a
BAFF antagonist or agonist, particularly where the antagonist or
agonist is specific for a human BAFF sequence or epitope. In
various embodiments, a humanized Baff protein expressed by a
non-human animal as described herein may comprise a variant amino
acid sequence. Variant human BAFF proteins having variations
associated with ligand binding residues have been reported. In
various embodiments, non-human animals of the present invention
express a humanized Baff protein variant. In various embodiments,
the variant is polymorphic at an amino acid position associated
with ligand binding. In various embodiments, non-human animals of
the present invention are used to determine the effect of ligand
binding through interaction with a polymorphic variant of human
BAFF. In some certain embodiments, non-human animals of the present
invention express a human BAFF splice variant protein that appears
in Table 3.
[0102] Cells from non-human animals of the present invention can be
isolated and used on an ad hoc basis, or can be maintained in
culture for many generations. For example, cells from non-human
animals of the present invention can be used in a variety of
cellular assays known in the art. In various embodiments, cells
from a non-human animal of the present invention are immortalized
and maintained in culture indefinitely (e.g., in serial
cultures).
[0103] In various embodiments, cells and/or non-human animals of
the present invention are used in a survival and/or proliferation
assay (e.g., employing B or T cells) to screen and develop
candidate therapeutics that modulate human BAFF. Survival of
auto-reactive B cells plays an important role in the chronic
pathology of autoimmune diseases, such as, for example, systemic
lupus erythematosus (SLE), therefore, candidate BAFF modulators
(e.g., antagonists) may be identified, characterized and developed
using cells of non-human animals of the present invention and/or a
non-human animal as described herein. In some embodiments, cells
and/or non-human animals of the present invention are used in a
survival assay to determine the number of antigen-specific plasma B
cells in the presence and absence of BAFF.
[0104] In various embodiments, cells and/or non-human animals of
the present invention are used in various immunization regimens to
determine the BAFF-mediated functions in the immune response to an
antigen. In some embodiments, candidate therapeutics that bind to,
or block one or more functions of, human BAFF are characterized in
a non-human animal of the present invention. Suitable measurements
include various cellular assays, proliferation assays, serum
immunoglobulin analysis (e.g., antibody titer), cytotoxicity
assays, characterization of ligand-receptor interactions
(immunoprecipitation assays). In some embodiments, non-human
animals of the present invention are used to characterize the
BAFF-mediated functions regulating an immune response to an
antigen. In some embodiments, the antigen is associated with an
autoimmune disease or condition. In some embodiments, the antigen
is a test antigen (e.g., ovalbumin or OVA). In some embodiments,
the antigen is a target associated with a disease or condition
suffered by one or more human patients in need of treatment.
[0105] In various embodiments, non-human animals of the present
invention are used in serum assays for determining titers of
double-stranded DNA (dsDNA) autoantibody production for testing the
pharmaco-toxicological aspects of candidate therapeutics that
target human BAFF. In some embodiments, double-stranded DNA (dsDNA)
autoantibody production in non-human animals of the present
invention results from one or more autoimmune diseases or
conditions induced in the non-human animal.
[0106] In various embodiments, cells and/or non-human animals of
the present invention are used to characterize the repertoire
and/or specificity of antibodies generated in an immune response to
antigen. In some embodiments, the immune response is characterized
by the generation of autoantibodies that are specific for one or
more tissues of a non-human animal of the present invention. In
some embodiments, the therapeutic potential of compounds or
biological agents to modulate BAFF-dependent regulation of the B
cell repertoire is characterized and/or developed in a non-human
animal of the present invention.
[0107] In various embodiments, non-human animals of the present
invention are used for challenge with one or more antigens to
determine the therapeutic potential of compounds or biological
agents to modulate BAFF-dependent regulation of an immune response,
including but not limited to, the specific T cell-dependent and B
cell-dependent responses to a given antigen.
[0108] In various embodiments, non-human animals of the present
invention are used in transplantation or adoptive transfer
experiments to determine the therapeutic potential of compounds or
biological agents to modulate BAFF-dependent regulation of new
lymphocytes and their immune function. In various embodiments,
non-human animals of the present invention are transplanted with
human B cells.
[0109] In various embodiments, cells of non-human animals of the
present invention are used to in T cell assays to determine the
therapeutic potential of compounds or biological agents to modulate
BAFF-dependent regulation of T cell-dependent response and
function. Exemplary T cell assays include, but are not limited to,
ELISpot, intracellular cytokine staining, major histocompatibility
complex (MHC) restriction, viral suppression assays, cytotoxicity
assays, proliferation assays and regulatory T cell suppression
assays.
[0110] In various embodiments, cells of non-human animals of the
present invention are used in tumor cell growth assays to determine
the therapeutic potential of compounds or biological agents to
modulate BAFF-dependent regulation and/or stimulation of tumor cell
growth.
[0111] In various embodiments, an autoimmune disease or condition
is induced in one or non-human animals of the present invention to
provide an in vivo system for determining the therapeutic potential
of compounds or biological agents to modulate BAFF-dependent
regulation of one or more functions of the autoimmune disease or
condition. In some embodiments, the autoimmune condition is an
inflammatory condition, for example, arthritis (e.g.,
collagen-induced arthritis, CIA).
[0112] Non-human animals of the present invention provide an in
vivo system for the analysis and testing of a drug or vaccine. In
various embodiments, a candidate drug or vaccine may be delivered
to one or more non-human animals of the present invention, followed
by monitoring of the non-human animals to determine one or more of
the immune response to the drug or vaccine, the safety profile of
the drug or vaccine, or the effect on a disease or condition.
Exemplary methods used to determine the safety profile include
measurements of toxicity, optimal dose concentration, efficacy of
the drug or vaccine, and possible risk factors. Such drugs or
vaccines may be improved and/or developed in such non-human
animals.
[0113] Non-human animals of the present invention provide an
improved in vivo system for the development and characterization of
candidate therapeutics for use in cancer. In various embodiments,
non-human animals of the present invention may be implanted with a
tumor, followed by administration of a candidate therapeutic. The
tumor may be allowed sufficient time to be established in one or
more locations within the non-human animal. Tumor cell
proliferation, growth, etc. may be measured both before and after
administration with the candidate therapeutic. Cytotoxicity of
candidate therapeutics may also be measured in the non-human animal
as desired.
[0114] Non-human animals of the present invention provide an
improved in vivo system for elucidating mechanisms of human
cell-to-cell interaction through adoptive transfer. In various
embodiments, non-human animals of the present invention may by
implanted with a tumor xenograft, followed by a second implantation
of tumor infiltrating lymphocytes in the non-human animals by
adoptive transfer to determine the effectiveness in eradication of
solid tumors or other malignancies. Such experiments may be done
with human cells (e.g., B cell lymphomas) due to the exclusive
presence of human BAFF without competition with endogenous Baff of
the non-human animal. Further, therapies and pharmaceuticals for
use in xenotransplantation can be improved and/or developed in such
non-human animals.
[0115] Non-human animals of the present invention provide an
improved in vivo system for maintenance and development of human
hematopoietic stem cells through engraftment. In various
embodiments, non-human animals of the present invention provide
improved development and maintenance of human stem cells within the
non-human animal. In various embodiments, increased populations of
differentiated human B and T cells are observed in the blood, bone
marrow, spleen and thymus of the non-human animal. In various
embodiments, non-human animals of the present invention provide an
increase in the level of engraftment of human hematopoietic stem
cells as compared to non-human animals that express both endogenous
non-human Baff and heterologous (e.g., human) BAFF.
[0116] Non-human animals of the present invention provide an
improved in vivo system for maintenance and development of human B
cells (e.g., from human donors) through engraftment. In various
embodiments, non-human animals of the present invention provide
improved development and maintenance of human B cells within the
non-human animal. In various embodiments, increased populations of
differentiated human B cells post-immunization are observed in one
or more of the blood, bone marrow, spleen or a lymph node of the
non-human animal. In various embodiments, non-human animals of the
present invention provide an increase in the level of engraftment
of human B cells as compared to non-human animals that express
endogenous non-human Baff.
EXAMPLES
[0117] The following examples are provided so as to describe to
those of ordinary skill in the art how to make and use methods and
compositions of the invention, and are not intended to limit the
scope of what the inventors regard as their invention. Unless
indicated otherwise, temperature is indicated in Celsius, and
pressure is at or near atmospheric.
Example 1
Humanization of an Endogenous Non-Human B-Cell Activating Factor
(Baff) Gene
[0118] This example illustrates exemplary methods of humanizing an
endogenous gene encoding B-cell Activating Factor (Baff) in a
non-human animal such as a rodent (e.g., a mouse). Human BAFF is
known to exist in several variant (or allelic) forms. The methods
described in this example can be employed to humanize an endogenous
Baff gene of a non-human animal using any human variant (or
allele), or combination of human variants (or alleles or fragments
thereof) as desired. In this example, a human BAFF gene that
appears in bacterial artificial chromosome (BAC) clone CTD-2355n18
is employed for humanizing an endogenous Baff gene of a mouse.
[0119] A targeting vector for humanization of an extracellular
region of a Baff gene was constructed using bacterial homologous
recombination and VELOCIGENE.RTM. technology (see, e.g., U.S. Pat.
No. 6,586,251 and Valenzuela et al., High-throughput engineering of
the mouse genome coupled with high-resolution expression analysis,
2003, Nature Biotech. 21(6):652-659). An exemplary process for
humanization of an endogenous Baff gene of a mouse is set forth in
FIG. 2.
[0120] Briefly, a human bacterial artificial chromosome (BAC) clone
CTD-2355n18 (Invitrogen) was modified to delete the 3' flanking
region of the human BAFF gene starting at approximately 206 bp 3'
of the human BAFF gene. The modification was performed by
homologous recombination in bacterial cells using a targeting
vector containing a self-deleting neomycin cassette flanked by
recombinase recognition sites (e.g., LoxP; see U.S. Pat. No.
8,354,389 and U.S. Pat. No. 8,518,392, both of which are herein
incorporated by reference) and a unique AsiSI restriction site
positioned at the 3' of the cassette. The resulting modified BAC
clone was modified in a second homologous recombination step in
bacterial cells using a spectinomycin cassette to delete sequence
5' of the human BAFF gene and exons 1, 2 and approximately 3146 bp
of intron 2. The spectinomycin cassette contained a unique I-CeuI
site 3' of the spectinomycin cassette. Therefore, the double
modified human BAC clone contained, from 5' to 3', a spectinomycin
cassette, an I-CeuI site, approximately 35,303 bp of human genomic
sequence containing most of human BAFF intron 2, human BAFF exons 3
to 6 and approximately 206 bp of human sequence 3' of human BAFF
exon 6, and a self-deleting neomycin cassette flanked by LoxP
sites, and a 3' AsiSI site.
[0121] Separately, a mouse BAC clone RP23-351L20 (Invitrogen) was
modified to specifically insert the modified human BAC clone
described above. In a first step, a hygromycin cassette flanked by
site-specific recombinase recognition sites (e.g., Frt) was used to
delete the sequence containing exons 3-6 and part of exon 7 of a
mouse Baff gene. The 3'UTR and polyadenylation signal was retained.
The hygromycin cassette included unique I-CeuI and AsiSI
restriction sites at flanking 5' and 3' ends, respectively.
Homologous recombination in bacterial cells with the hygromycin
cassette resulted in a .about.25,148 bp deletion in the mouse Baff
gene corresponding to exons 3-7, leaving intact .about.3069 bp of
the mouse Baff intron 2. The 3' end of the hygromycin cassette was
targeted to approximately the middle of the 3' UTR of the mouse
Baff gene (of exon 7) in BAC clone RP23-351L20. The modified mouse
BAC clone having a deletion of mouse Baff exons 3-6 and 7 (in part)
from homologous recombination with the hygromycin cassette was
modified in a second step using the modified human BAC clone having
a deletion of human BAFF exons 1-2 and .about.3146 bp of intron 2.
This was achieved by through the unique restriction enzyme sites
common between the two modified BAC clones. Each modified BAC clone
was digested with I-CeuI and AsiSI to produce compatible cohesive
fragments (FIG. 2). The final targeting vector, made by ligation of
the compatible restriction fragments, contained, from 5' to 3',
mouse genomic sequence containing mouse Lig4 and Abdh13 genes,
.about.14.5 kb of mouse genomic sequence, exons 1 and 2 of a mouse
Baff gene, .about.3069 by of intron 2 of a mouse Baff gene, an
I-CeuI site, .about.35.3 kb of human genomic sequence containing
exons 3 to 6 of a human BAFF gene, a self-deleting neomycin
cassette flanked by recombinase recognition sites, an AsiSI site,
part of a mouse Baff exon 7 that included a 3'UTR and
polyadenylation signal, and mouse genomic sequence 3' of a mouse
Baff gene.
[0122] The final targeting vector was used to electroporate
BALB-Rag2.sup.-/-IL2R.gamma..sub.C.sup.-/- (DKO) mouse embryonic
stem (ES) cells to create modified ES cells comprising a Baff gene
at an endogenous Baff locus that is humanized from approximately
the middle of intron 2 of a mouse Baff gene (.about.3000 bp 3' of
splice donor site) to approximately 100 bp 3' of the
polyadenylation site of a human BAFF gene that was inserted into
approximately the middle of the 3'UTR of a mouse Baff gene (FIG.
1). Positively targeted ES cells containing a humanized BAFF gene
were identified by an assay (Valenzuela et al., supra) that
detected the presence of the human BAFF sequence and confirmed loss
of mouse Baff sequences. Table 4 sets forth the primers and probes
that were used to confirm humanization of an endogenous Baff gene
as described above. hBAFF: human BAFF; mBaff: mouse Baff.
[0123] Positive ES cell clones were then used to implant female
mice using the VELOCIMOUSE.RTM. method (see, e.g., U.S. Pat. No.
7,294,754 and Poueymirou et al., F0 generation mice that are
essentially fully derived from the donor gene-targeted ES cells
allowing immediate phenotypic analyses, 2007, Nature Biotech.
25(1):91-99) to generate a litter of pups containing an insertion
of exons 3 to 6 of a human BAFF gene into an endogenous Baff gene
of a mouse. Mice bearing the humanization of exons 3 to 6 of an
endogenous Baff gene were again confirmed identified by genotyping
of DNA isolated from tail snips using a modification of allele
assay (Valenzuela et al., supra) that detected the presence of the
human BAFF gene sequences. Pups are genotyped and cohorts of
animals heterozygous for the humanized Baff gene construct are
selected for characterization.
TABLE-US-00004 TABLE 4 Name Location Primer Sequence (5'-3')
mBaff-1 mBaff intron 2 Forward GGACAGCAGATAGGAAAGCTTCTTG SEQ ID NO:
8 Reverse GGGACGGACACTCATTTGAC SEQ ID NO: 9 Probe
TAGGAATCCCAGTCCTTAGAACCGCA SEQ ID NO: 10 mBaff-2 mBaff exon 7
Forward CCTCGGGAGAATGCACAGAT SEQ ID NO: 11 Reverse
GCACTCCAGCAAGTGAGTTAC SEQ ID NO: 12 Probe TCACGCAACGGAGACGACACCTT
SEQ ID NO: 13 hBAFF-1 hBAFF intron 2 Forward CCGGTTGGCATTTCTGGCTTAG
SEQ ID NO: 14 Reverse GGCTGGATGGTCAAGTTCTACA SEQ ID NO: 15 Probe
TTCCAGGCTGTAACATGAGTGTTGGA SEQ ID NO: 16 hBAFF-2 hBAFF intron 5
Forward ACACCAGACAGGTGACTTAGGAA SEQ ID NO: 17 Reverse
GCTCCTGGGTGCAAAGGTA SEQ ID NO: 18 Probe TGCGAAAGTGTAGGCGCAAACC SEQ
ID NO: 19
Equivalents
[0124] Having thus described several aspects of at least one
embodiment of this invention, it is to be appreciated by those
skilled in the art that various alterations, modifications, and
improvements will readily occur to those skilled in the art. Such
alterations, modifications, and improvements are intended to be
part of this disclosure, and are intended to be within the spirit
and scope of the invention. Accordingly, the foregoing description
and drawing are by way of example only and the invention is
described in detail by the claims that follow.
[0125] Use of ordinal terms such as "first," "second," "third,"
etc., in the claims to modify a claim element does not by itself
connote any priority, precedence, or order of one claim element
over another or the temporal order in which acts of a method are
performed, but are used merely as labels to distinguish one claim
element having a certain name from another element having a same
name (but for use of the ordinal term) to distinguish the claim
elements.
[0126] The articles "a" and "an" as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to include the plural referents.
Claims or descriptions that include "or" between one or more
members of a group are considered satisfied if one, more than one,
or all of the group members are present in, employed in, or
otherwise relevant to a given product or process unless indicated
to the contrary or otherwise evident from the context. The
invention includes embodiments in which exactly one member of the
group is present in, employed in, or otherwise relevant to a given
product or process. The invention also includes embodiments in
which more than one, or the entire group members are present in,
employed in, or otherwise relevant to a given product or process.
Furthermore, it is to be understood that the invention encompasses
all variations, combinations, and permutations in which one or more
limitations, elements, clauses, descriptive terms, etc., from one
or more of the listed claims is introduced into another claim
dependent on the same base claim (or, as relevant, any other claim)
unless otherwise indicated or unless it would be evident to one of
ordinary skill in the art that a contradiction or inconsistency
would arise. Where elements are presented as lists, (e.g., in
Markush group or similar format) it is to be understood that each
subgroup of the elements is also disclosed, and any element(s) can
be removed from the group. It should be understood that, in
general, where the invention, or aspects of the invention, is/are
referred to as comprising particular elements, features, etc.,
certain embodiments of the invention or aspects of the invention
consist, or consist essentially of, such elements, features, etc.
For purposes of simplicity those embodiments have not in every case
been specifically set forth in so many words herein. It should also
be understood that any embodiment or aspect of the invention can be
explicitly excluded from the claims, regardless of whether the
specific exclusion is recited in the specification.
[0127] Those skilled in the art will appreciate typical standards
of deviation or error attributable to values obtained in assays or
other processes described herein.
[0128] The publications, websites and other reference materials
referenced herein to describe the background of the invention and
to provide additional detail regarding its practice are hereby
incorporated by reference.
Sequence CWU 1
1
1911710DNAMus musculus 1ggcacgaggc agattgagca atccatggaa ggccagagcc
agagaaccta cttcagggta 60gcaaaagatg cagaagaaag tcaggagagc gctcctgggg
gaacccagcc ctgccatgct 120ctgagggcag tctcccagga cacagatgac
aggaaatgac ccacccctgt ggtcacttac 180tccaaaggcc tagaccttca
aagtgctcct cgtggaatgg atgagtctgc aaagaccctg 240ccaccaccgt
gcctctgttt ttgctccgag aaaggagaag atatgaaagt gggatatgat
300cccatcactc cgcagaagga ggagggtgcc tggtttggga tctgcaggga
tggaaggctg 360ctggctgcta ccctcctgct ggccctgttg tccagcagtt
tcacagcgat gtccttgtac 420cagttggctg ccttgcaagc agacctgatg
aacctgcgca tggagctgca gagctaccga 480ggttcagcaa caccagccgc
cgcgggtgct ccagagttga ccgctggagt caaactcctg 540acaccggcag
ctcctcgacc ccacaactcc agccgcggcc acaggaacag acgcgctttc
600cagggaccag aggaaacaga acaagatgta gacctctcag ctcctcctgc
accatgcctg 660cctggatgcc gccattctca acatgatgat aatggaatga
acctcagaaa catcattcaa 720gactgtctgc agctgattgc agacagcgac
acgccgacta tacgaaaagg aacttacaca 780tttgttccat ggcttctcag
ctttaaaaga ggaaatgcct tggaggagaa agagaacaaa 840atagtggtga
ggcaaacagg ctatttcttc atctacagcc aggttctata cacggacccc
900atctttgcta tgggtcatgt catccagagg aagaaagtac acgtctttgg
ggacgagctg 960agcctggtga ccctgttccg atgtattcag aatatgccca
aaacactgcc caacaattcc 1020tgctactcgg ctggcatcgc gaggctggaa
gaaggagatg agattcagct tgcaattcct 1080cgggagaatg cacagatttc
acgcaacgga gacgacacct tctttggtgc cctaaaactg 1140ctgtaactca
cttgctggag tgcgtgatcc ccttccctcg tcttctctgt acctccgagg
1200gagaaacaga cgactggaaa aactaaaaga tggggaaagc cgtcagcgaa
agttttctcg 1260tgacccgttg aatctgatcc aaaccaggaa atataacaga
cagccacaac cgaagtgtgc 1320catgtgagtt atgagaaacg gagcccgcgc
tcagaaagac cggatgagga agaccgtttt 1380ctccagtcct ttgccaacac
gcaccgcaac cttgcttttt gccttgggtg acacatgttc 1440agaatgcagg
gagatttcct tgttttgcga tttgccatga gaagagggcc cacaactgca
1500ggtcactgaa gcattcacgc taagtctcag gatttactct cccttctcat
gctaagtaca 1560cacacgctct tttccaggta atactatggg atactatgga
aaggttgttt gtttttaaat 1620ctagaagtct tgaactggca atagacaaaa
atccttataa attcaagtgt aaaataaact 1680taattaaaaa ggtttaagtg
tgaaaaaaaa 17102309PRTMus musculus 2Met Asp Glu Ser Ala Lys Thr Leu
Pro Pro Pro Cys Leu Cys Phe Cys 1 5 10 15 Ser Glu Lys Gly Glu Asp
Met Lys Val Gly Tyr Asp Pro Ile Thr Pro 20 25 30 Gln Lys Glu Glu
Gly Ala Trp Phe Gly Ile Cys Arg Asp Gly Arg Leu 35 40 45 Leu Ala
Ala Thr Leu Leu Leu Ala Leu Leu Ser Ser Ser Phe Thr Ala 50 55 60
Met Ser Leu Tyr Gln Leu Ala Ala Leu Gln Ala Asp Leu Met Asn Leu 65
70 75 80 Arg Met Glu Leu Gln Ser Tyr Arg Gly Ser Ala Thr Pro Ala
Ala Ala 85 90 95 Gly Ala Pro Glu Leu Thr Ala Gly Val Lys Leu Leu
Thr Pro Ala Ala 100 105 110 Pro Arg Pro His Asn Ser Ser Arg Gly His
Arg Asn Arg Arg Ala Phe 115 120 125 Gln Gly Pro Glu Glu Thr Glu Gln
Asp Val Asp Leu Ser Ala Pro Pro 130 135 140 Ala Pro Cys Leu Pro Gly
Cys Arg His Ser Gln His Asp Asp Asn Gly 145 150 155 160 Met Asn Leu
Arg Asn Ile Ile Gln Asp Cys Leu Gln Leu Ile Ala Asp 165 170 175 Ser
Asp Thr Pro Thr Ile Arg Lys Gly Thr Tyr Thr Phe Val Pro Trp 180 185
190 Leu Leu Ser Phe Lys Arg Gly Asn Ala Leu Glu Glu Lys Glu Asn Lys
195 200 205 Ile Val Val Arg Gln Thr Gly Tyr Phe Phe Ile Tyr Ser Gln
Val Leu 210 215 220 Tyr Thr Asp Pro Ile Phe Ala Met Gly His Val Ile
Gln Arg Lys Lys 225 230 235 240 Val His Val Phe Gly Asp Glu Leu Ser
Leu Val Thr Leu Phe Arg Cys 245 250 255 Ile Gln Asn Met Pro Lys Thr
Leu Pro Asn Asn Ser Cys Tyr Ser Ala 260 265 270 Gly Ile Ala Arg Leu
Glu Glu Gly Asp Glu Ile Gln Leu Ala Ile Pro 275 280 285 Arg Glu Asn
Ala Gln Ile Ser Arg Asn Gly Asp Asp Thr Phe Phe Gly 290 295 300 Ala
Leu Lys Leu Leu 305 3290PRTMus musculus 3Met Asp Glu Ser Ala Lys
Thr Leu Pro Pro Pro Cys Leu Cys Phe Cys 1 5 10 15 Ser Glu Lys Gly
Glu Asp Met Lys Val Gly Tyr Asp Pro Ile Thr Pro 20 25 30 Gln Lys
Glu Glu Gly Ala Trp Phe Gly Ile Cys Arg Asp Gly Arg Leu 35 40 45
Leu Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Ser Ser Phe Thr Ala 50
55 60 Met Ser Leu Tyr Gln Leu Ala Ala Leu Gln Ala Asp Leu Met Asn
Leu 65 70 75 80 Arg Met Glu Leu Gln Ser Tyr Arg Gly Ser Ala Thr Pro
Ala Ala Ala 85 90 95 Gly Ala Pro Glu Leu Thr Ala Gly Val Lys Leu
Leu Thr Pro Ala Ala 100 105 110 Pro Arg Pro His Asn Ser Ser Arg Gly
His Arg Asn Arg Arg Ala Phe 115 120 125 Gln Gly Pro Glu Glu Thr Glu
Gln Asp Val Asp Leu Ser Ala Pro Pro 130 135 140 Ala Pro Cys Leu Pro
Gly Cys Arg His Ser Gln His Asp Asp Asn Gly 145 150 155 160 Met Asn
Leu Arg Asn Arg Thr Tyr Thr Phe Val Pro Trp Leu Leu Ser 165 170 175
Phe Lys Arg Gly Asn Ala Leu Glu Glu Lys Glu Asn Lys Ile Val Val 180
185 190 Arg Gln Thr Gly Tyr Phe Phe Ile Tyr Ser Gln Val Leu Tyr Thr
Asp 195 200 205 Pro Ile Phe Ala Met Gly His Val Ile Gln Arg Lys Lys
Val His Val 210 215 220 Phe Gly Asp Glu Leu Ser Leu Val Thr Leu Phe
Arg Cys Ile Gln Asn 225 230 235 240 Met Pro Lys Thr Leu Pro Asn Asn
Ser Cys Tyr Ser Ala Gly Ile Ala 245 250 255 Arg Leu Glu Glu Gly Asp
Glu Ile Gln Leu Ala Ile Pro Arg Glu Asn 260 265 270 Ala Gln Ile Ser
Arg Asn Gly Asp Asp Thr Phe Phe Gly Ala Leu Lys 275 280 285 Leu Leu
290 42675DNAHomo sapiens 4gaaattctta caaaaactga aagtgaaatg
aggaagacag attgagcaat ccaatcggag 60ggtaaatgcc agcaaaccta ctgtacagta
ggggtagaga tgcagaaagg cagaaaggag 120aaaattcagg ataactctcc
tgaggggtga gccaagccct gccatgtagt gcacgcagga 180catcaacaaa
cacagataac aggaaatgat ccattccctg tggtcactta ttctaaaggc
240cccaaccttc aaagttcaag tagtgatatg gatgactcca cagaaaggga
gcagtcacgc 300cttacttctt gccttaagaa aagagaagaa atgaaactga
aggagtgtgt ttccatcctc 360ccacggaagg aaagcccctc tgtccgatcc
tccaaagacg gaaagctgct ggctgcaacc 420ttgctgctgg cactgctgtc
ttgctgcctc acggtggtgt ctttctacca ggtggccgcc 480ctgcaagggg
acctggccag cctccgggca gagctgcagg gccaccacgc ggagaagctg
540ccagcaggag caggagcccc caaggccggc ctggaggaag ctccagctgt
caccgcggga 600ctgaaaatct ttgaaccacc agctccagga gaaggcaact
ccagtcagaa cagcagaaat 660aagcgtgccg ttcagggtcc agaagaaaca
gtcactcaag actgcttgca actgattgca 720gacagtgaaa caccaactat
acaaaaagga tcttacacat ttgttccatg gcttctcagc 780tttaaaaggg
gaagtgccct agaagaaaaa gagaataaaa tattggtcaa agaaactggt
840tactttttta tatatggtca ggttttatat actgataaga cctacgccat
gggacatcta 900attcagagga agaaggtcca tgtctttggg gatgaattga
gtctggtgac tttgtttcga 960tgtattcaaa atatgcctga aacactaccc
aataattcct gctattcagc tggcattgca 1020aaactggaag aaggagatga
actccaactt gcaataccaa gagaaaatgc acaaatatca 1080ctggatggag
atgtcacatt ttttggtgca ttgaaactgc tgtgacctac ttacaccatg
1140tctgtagcta ttttcctccc tttctctgta cctctaagaa gaaagaatct
aactgaaaat 1200accaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaagtagtt
accattgcct tttctgtgag 1260ctatttgttt tggtttgctg aaactagtcc
aaaacaggaa atttaacaga cagccacagc 1320caaagagtgt catgtgaatt
acaagaaata gagcccattt agggaaagat agaactagaa 1380aggcttttca
ttataattcc atgttgaaca attgagtcat agcttcttat cttggaggaa
1440ggacacaatt caaaggggca gtaaggattt tgtaaaacgt ggcatccata
atttactatg 1500gagcaagtgc ccacatctct aggacattaa gacatttatg
agaaatctca ggattcatct 1560tctgttttta tgttaaatgc actccctcct
tttcagttaa cattataaaa agtaaaaaat 1620gaaaatttta gaaatcttgc
attagacaca tgaaaaaata actaaaagtt taaatttaaa 1680tatgaaacaa
ttttgctgaa aatagtatcc atatactatt taagtctttt atggttattt
1740caagtataca atttctatct gtaatgtaat atattaccca cacatttttt
tcacaggaga 1800gagagaatat cctcatttgt ttatgctcat gtgtattttc
tatagtgaat ttcagaaact 1860tttaatatca ggtaatttca atttatgcct
ataaagcatt gattgaaaaa taactagaat 1920tgtgcatata taacacataa
tctccaacag aagttactga atacattcat actaatgtaa 1980tgtaatttcc
ctttatttct tgctcttctg tttcaaactg ctgctattgt agtttacata
2040tcccaacctt taaaaatatt cctcttatta gctttatatt cactttatag
aagttgagtt 2100ttaattaaaa ttcttggcat cctgaagtat gtcacatagc
atgtgctcct tataaatatg 2160ttgatatctc agaagacagc atcccggttt
tcattttata aagtaccata cttaagaatg 2220ctgtaatact tatcttttat
aacatgtttc cttcgctttg cttgtctttt atgtcatcag 2280ttttaactgt
ttacttcatt taacagttta catcattcaa cagtttactt cattaaacag
2340taggtggaaa aatagatgcc agtctatgaa aatcttccca tctatatcaa
aatacttttc 2400aaggatatac ttttcaaaac aaacgattta aattttatgt
ttaaaatata aactttagat 2460ttaaacttta tttaaatatc tggttcctat
gattttgact tcagtaagtt caaataaaat 2520atattttgca attcattttt
acattataat ttaaaaagaa gaagcgataa gtggagtcag 2580tttcaatgct
aggtggggtg gttaatgatt tttctggtgt tgctgctaat gtggattaac
2640aaataaaaac attcattgcc ttttgcctca taaaa 26755285PRTHomo sapiens
5Met Asp Asp Ser Thr Glu Arg Glu Gln Ser Arg Leu Thr Ser Cys Leu 1
5 10 15 Lys Lys Arg Glu Glu Met Lys Leu Lys Glu Cys Val Ser Ile Leu
Pro 20 25 30 Arg Lys Glu Ser Pro Ser Val Arg Ser Ser Lys Asp Gly
Lys Leu Leu 35 40 45 Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Cys
Cys Leu Thr Val Val 50 55 60 Ser Phe Tyr Gln Val Ala Ala Leu Gln
Gly Asp Leu Ala Ser Leu Arg 65 70 75 80 Ala Glu Leu Gln Gly His His
Ala Glu Lys Leu Pro Ala Gly Ala Gly 85 90 95 Ala Pro Lys Ala Gly
Leu Glu Glu Ala Pro Ala Val Thr Ala Gly Leu 100 105 110 Lys Ile Phe
Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Asn 115 120 125 Ser
Arg Asn Lys Arg Ala Val Gln Gly Pro Glu Glu Thr Val Thr Gln 130 135
140 Asp Cys Leu Gln Leu Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys
145 150 155 160 Gly Ser Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys
Arg Gly Ser 165 170 175 Ala Leu Glu Glu Lys Glu Asn Lys Ile Leu Val
Lys Glu Thr Gly Tyr 180 185 190 Phe Phe Ile Tyr Gly Gln Val Leu Tyr
Thr Asp Lys Thr Tyr Ala Met 195 200 205 Gly His Leu Ile Gln Arg Lys
Lys Val His Val Phe Gly Asp Glu Leu 210 215 220 Ser Leu Val Thr Leu
Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu 225 230 235 240 Pro Asn
Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly 245 250 255
Asp Glu Leu Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu 260
265 270 Asp Gly Asp Val Thr Phe Phe Gly Ala Leu Lys Leu Leu 275 280
285 6266PRTHomo sapiens 6Met Asp Asp Ser Thr Glu Arg Glu Gln Ser
Arg Leu Thr Ser Cys Leu 1 5 10 15 Lys Lys Arg Glu Glu Met Lys Leu
Lys Glu Cys Val Ser Ile Leu Pro 20 25 30 Arg Lys Glu Ser Pro Ser
Val Arg Ser Ser Lys Asp Gly Lys Leu Leu 35 40 45 Ala Ala Thr Leu
Leu Leu Ala Leu Leu Ser Cys Cys Leu Thr Val Val 50 55 60 Ser Phe
Tyr Gln Val Ala Ala Leu Gln Gly Asp Leu Ala Ser Leu Arg 65 70 75 80
Ala Glu Leu Gln Gly His His Ala Glu Lys Leu Pro Ala Gly Ala Gly 85
90 95 Ala Pro Lys Ala Gly Leu Glu Glu Ala Pro Ala Val Thr Ala Gly
Leu 100 105 110 Lys Ile Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser
Ser Gln Asn 115 120 125 Ser Arg Asn Lys Arg Ala Val Gln Gly Pro Glu
Glu Thr Gly Ser Tyr 130 135 140 Thr Phe Val Pro Trp Leu Leu Ser Phe
Lys Arg Gly Ser Ala Leu Glu 145 150 155 160 Glu Lys Glu Asn Lys Ile
Leu Val Lys Glu Thr Gly Tyr Phe Phe Ile 165 170 175 Tyr Gly Gln Val
Leu Tyr Thr Asp Lys Thr Tyr Ala Met Gly His Leu 180 185 190 Ile Gln
Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu Val 195 200 205
Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu Pro Asn Asn 210
215 220 Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp Glu
Leu 225 230 235 240 Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser
Leu Asp Gly Asp 245 250 255 Val Thr Phe Phe Gly Ala Leu Lys Leu Leu
260 265 7278PRTArtificial SequenceHumanized BAFF Protein 7Met Asp
Glu Ser Ala Lys Thr Leu Pro Pro Pro Cys Leu Cys Phe Cys 1 5 10 15
Ser Glu Lys Gly Glu Asp Met Lys Val Gly Tyr Asp Pro Ile Thr Pro 20
25 30 Gln Lys Glu Glu Gly Ala Trp Phe Gly Ile Cys Arg Asp Gly Arg
Leu 35 40 45 Leu Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Ser Ser
Phe Thr Ala 50 55 60 Met Ser Leu Tyr Gln Leu Ala Ala Leu Gln Ala
Asp Leu Met Asn Leu 65 70 75 80 Arg Met Glu Leu Gln Ser Tyr Arg Gly
Ser Ala Thr Pro Ala Ala Ala 85 90 95 Gly Ala Pro Glu Leu Thr Ala
Gly Val Lys Leu Leu Thr Pro Ala Ala 100 105 110 Pro Arg Pro His Asn
Ser Ser Arg Gly His Arg Asn Arg Arg Ala Phe 115 120 125 Gln Gly Pro
Glu Glu Thr Val Thr Gln Asp Cys Leu Gln Leu Ile Ala 130 135 140 Asp
Ser Glu Thr Pro Thr Ile Gln Lys Gly Ser Tyr Thr Phe Val Pro 145 150
155 160 Trp Leu Leu Ser Phe Lys Arg Gly Ser Ala Leu Glu Glu Lys Glu
Asn 165 170 175 Lys Ile Leu Val Lys Glu Thr Gly Tyr Phe Phe Ile Tyr
Gly Gln Val 180 185 190 Leu Tyr Thr Asp Lys Thr Tyr Ala Met Gly His
Leu Ile Gln Arg Lys 195 200 205 Lys Val His Val Phe Gly Asp Glu Leu
Ser Leu Val Thr Leu Phe Arg 210 215 220 Cys Ile Gln Asn Met Pro Glu
Thr Leu Pro Asn Asn Ser Cys Tyr Ser 225 230 235 240 Ala Gly Ile Ala
Lys Leu Glu Glu Gly Asp Glu Leu Gln Leu Ala Ile 245 250 255 Pro Arg
Glu Asn Ala Gln Ile Ser Leu Asp Gly Asp Val Thr Phe Phe 260 265 270
Gly Ala Leu Lys Leu Leu 275 825DNAArtificial SequenceSynthetic
Oligonucleotide mBaff-1, mBaff intron 2, Forward 8ggacagcaga
taggaaagct tcttg 25920DNAArtificial SequenceSynthetic
Oligonucleotide mBaff-1, mBaff intron 2, Reverse 9gggacggaca
ctcatttgac 201026DNAArtificial SequenceSynthetic Oligonucleotide
mBaff-1, mBaff intron 2, Probe 10taggaatccc agtccttaga accgca
261120DNAArtificial SequenceSynthetic Oligonucleotide mBaff-2,
mBaff exon 7, Forward 11cctcgggaga atgcacagat 201221DNAArtificial
SequenceSynthetic Oligonucleotide mBaff-2, mBaff exon 7, Reverse
12gcactccagc aagtgagtta c 211323DNAArtificial SequenceSynthetic
Oligonucleotide mBaff-2, mBaff exon 7, Probe 13tcacgcaacg
gagacgacac ctt 231422DNAArtificial SequenceSynthetic
Oligonucleotide hBaff-1, hBAFF intron 2, Forward 14ccggttggca
tttctggctt ag 221522DNAArtificial SequenceSynthetic Oligonucleotide
hBaff-1, hBAFF intron 2, Reverse 15ggctggatgg tcaagttcta ca
221626DNAArtificial SequenceSynthetic Oligonucleotide hBaff-1,
hBAFF intron 2, Probe 16ttccaggctg taacatgagt gttgga
261723DNAArtificial SequenceSynthetic Oligonucleotide hBaff-2,
hBAFF intron 5, Forward 17acaccagaca ggtgacttag gaa
231819DNAArtificial SequenceSynthetic Oligonucleotide hBaff-2,
hBAFF intron 5, Reverse 18gctcctgggt gcaaaggta 191922DNAArtificial
SequenceSynthetic Oligonucleotide hBaff-2, hBAFF intron 5, Probe
19tgcgaaagtg taggcgcaaa cc 22
* * * * *