Final Report PBG(2)

8/6/2019 Final Report PBG(2)

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Penn Biotechnology Group

Roy Amariglio

Daniel Chen

Daniel Hussey

Santhosh Palani

Jason Ruth

Najaf A. Shah

Jonathan Zucker

OPPORTUNITIES FOR

BIONANOMATRIX IN THE

GENETIC TESTING

MARKET


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OPPORTUNITIES FOR BIONANOMATRIX IN

THE GENETIC TESTING MARKET

Contents

Prenatal Genetic testing ................................................................................................. 3

Background .................................................................................................................. 3

Table 1. Pregnancy distribution in the United States and European Union. ...... 3

Karyotyping.................................................................................................................. 4

FISH ............................................................................................................................. 4

Screening...................................................................................................................... 5

Future trends ............................................................................................................ 6

Table 2. Selected companies developing noninvasive prenatal genetic

diagnostics methods. (Adapted from IN VIVO, Vol. 27, No. 10, Nov. 2009). ........ 7

Scientific Exploration...................................................................................................... 8

Overview of chromosomal abnormalities ................................................................... 8

Down Syndrome ....................................................................................................... 8

Sex chromosome aneuploidies ................................................................................. 9

Neural tube defects (NTDs) ..................................................................................... 9

Cardiac chromosomal abnormalities....................................................................... 9

Deletions................................................................................................................. 10

Potential application areas ....................................................................................... 10

Table 3. Disorders associated with aneuploidy .................................................... 11

Table 4. Disorders associated with large chromosomal deletions ....................... 11

Table 5. Disorders associated with gene deletions ............................................... 12

Diseases and their corresponding genetic loci ...................................................... 12

Intellectual Property ..................................................................................................... 17

Potential issues.......................................................................................................... 17

Market Analysis ............................................................................................................ 19


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Table 7. Global market for gene/chromosome diagnostics by segment (Source

BCC)........................................................................................................................ 20

Table 8. Demand for DNA microarray by end use. .............................................. 21

Forces driving growth in the DNA microarray market ........................................... 21

Table 9. Market distribution for molecular diagnostics for inherited genetic

disease testing in the US, ($ millions, estimates). ............................................... 22

Table 10. Market driving forces. ........................................................................... 22

Competitive Analysis .................................................................................................... 23

Cost per use (reagents, labor/technical staff requirements, time additional per

run).......................................................................................................................... 23

Advantages, limitations, efforts to improve that currently used technology...... 23

Table 11. Summary Table of Competitive Technologies...................................... 25

Risks of addressing this market at this time........................................................ 25

Cancer Genetic Testing................................................................................................. 27

Background ................................................................................................................ 27

All cancers .................................................................................................................. 27

Leukemia.................................................................................................................... 27

Colorectal cancer........................................................................................................ 28

Breast cancer ............................................................................................................. 30

Table 12. Currently available cancer genetic tests. ............................................ 31

Bibliography .................................................................................................................. 33


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PRENATAL GENETIC TESTING

Background Annually, there are roughly 6 million pregnancies in the United States,

approximately 4 million of which result in live births, and the remaining 2 million

ending in pregnancy losses (1), with induced abortion and fetal losses each

contributing approximately 1 million (2). Among the overall population of pregnant

women, approximately 23% are above the age of 30 (225 in 1000, year 2004), and

approximately 9% are over the age of 35 (93 in 1000, year 2004) (2).

Table 1. Pregnancy distribution in the United States and

European Union.

To check for chromosomal abnormalities (of which Down syndrome is most common),women over the age of 35 are offered amniocentesis, which is performed at 15-20

weeks. Women who have previously experienced pregnancies with chromosomal or

other genetic abnormalities are also offered this test. The amniocentesis procedure

involves the use of a needle guided by ultrasound to withdraw 1-2 tbsp of amniotic

fluid from which fetal cells are separated and cultured in the lab for 10-12 days, in

order to perform chromosomal and genetic analyses. Amniotic fluid is also used to

look for aberrant expression of various proteins, which may be indicative of disease

US EU

Total Pregnancies 6,000,000 7,300,000

Induced Abortion 1,000,000 1216667

Fetal loses 1,000,000 1216667

Live birth 4,000,000 4866667

Less than 30 3,100,000 3,309,334

Above 30 900,000 1119333

Above 35 372,000 438000


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(3). The amniocentesis procedure carries a risk of miscarriage, which is estimated to

between 1-2 out of 400 procedures performed; in contrast, chorionic villus sampling

(CVS), is typically performed at 10-12 weeks, carries a miscarriage risk of roughly

1% (1). In the United States, amniocentesis is performed roughly 200,000 times a

year; this may be a good estimate of the size of the prenatal genetic testing market.

KaryotypingTo screen for chromosomal aberrations, the fluid obtained via amniocentesis is

analyzed using karyotyping, a low-resolution (detection of changes larger than 5MB)

method that has been the de-facto standard, and which is now being increasingly

supplanted by higher resolution technologies. A quasi-reliable reference suggests

that approximately 400,000 karyotypes are performed each year in the United

States and Canada (this number seems plausible with respect to the number of

amniocentesis procedures performed per year; however, karyotyping is the standard

analysis method for a number of other genetic disorders. Although low in resolution,

karyotyping remains a reliable method for detecting aneuploidy, and other gross

chromosomal aberrations such as breaks, fusions, and rearrangements. The cost

associated with performing a karyotype test mainly involves the cost of technician

labor since reagent and other associated costs are relatively low.

FISH

Fluorescence in situ hybridization (FISH) is often used to provide rapid and fairly

accurate determination of chromosomal aberrations. FISH uses chromosome-

specific DNA probes and is performed when cells are in metaphase and

chromosomes are condensed and can be individually distinguished.


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Screening

In the past decade, prenatal screening has been introduced to screen pregnancies,

prior to invasive prenatal genetic diagnosis (i.e. amniocentesis or CVS), in order to

both reduce the number of unnecessary invasive procedures, and expand the age

range of women who are able to reasonably receive invasive prenatal diagnosis. The

consequence of the success of screening has been a precipitous drop in the number ofgenetic prenatal diagnoses. In 2002, amniocentesis was performed in 1.9% of

pregnancies, while in 2003 it was performed in 1.7% of pregnancies (1). In 2007, The

American College of Obstetricians and Gynecologists (ACOG) published new

guidelines for prenatal screening which recommend that age not be the primary

screening technique, and that combined ultrasonography and serum screening

methods be used (4). Likely due in large part to this report, our research indicates

that the level of amniocentesis performed may have fallen by 80%, down to 0.36% of

all pregnancies [primary research].

Genetic diagnoses with amniocentesis or CVS are currently performed in either

pregnancies with Down Syndrome which have undergone successful prenatal

screening, or non-Down Syndrome pregnancies which have resulted in false positive

screening results. In 2007 when the new ACOG guidelines were published, a 5%

false positive rate was considered acceptable (4). In 2009, false positive rates for

screening tests at 2.29% were considered acceptable (5). As the false positive rate is

related to the number of invasive screenings performed and thereby the number of

procedure-related fetus losses in normal pregnancies, great effort is underway to

decrease the false positive rate while maintaining a high (> 90%) true positive rate,

which would further decrease the number of genetic diagnoses performed in the

future (5).

*Projected trend for 2009.


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Future trendsIn the future, noninvasive methods of detecting fetal DNA, such as either nucleated

fetal erythrocytes or fetal DNA in maternal blood, may essentially eliminate the risk

of attaining fetal DNA. If this occurs, then it is probable that genetic methods

currently used for prenatal diagnosis will obviate serum marker screening due to

their higher sensitivity and specificity. This assumes that:

1. The sensitivity and specificity remain high with the new sources of

DNA (i.e. assuming that sufficient DNA is attainable by these noninvasivemethods to maintain a sensitivity and specificity with genetic testing which

are equivalent to the sensitivity and specificity provided by the current

amount of DNA from invasive testing)

2. This is a more cost effective method than the current screening method,

from the standpoint of public health.

While circulating fetal DNA is an area of current interest, and epigenetic markers

are being used to distinguish fetal from paternal DNA, this DNA is fractionated, and

therefore not ideal for the BNM platform. Fetal nucleated red blood cells (fNRBCs),

however, are a promising noninvasive source of the entire genetic sequence of afetus, which would be suitable for BNMs platform. Though nucleated fetal

progenitor cells can persist for long periods after pregnancy (6), NRBCs have a

limited life span and therefore only represent a current pregnancy (7). Additionally,

a promising new publication reveals that spectral scattering characteristics can be

used to reliably differentiate fNRVCs from adult NRBCs, using only the endogenous

properties of the microstructure of the cells and thereby not requiring any additional

imaging contrast agents (8). While not currently used, there is movement in clinical

research to refine maternal blood methods to allow for noninvasive screening, such

as the NIH funded multicenter NIFTY trial (9). fNRBCs occur at a rate of 1 in 109

maternal blood cells. Regarding our finding that fetal nucleated cells areidentifiable in the cervical mucus plug which develops during pregnancy, other than

these cells and fetal DNA in maternal blood there are likely no other sources of fetal

DNA available in a manner less invasive than amniocentesis or CVS (10). No

research has been published which regards to attaining fetal DNA from cervical

mucus plugs since 2006.


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It is expected that the non-invasive methods for harvesting fetal DNA that are

currently in development will yield smaller sample size than amniocentisis and

CVS. It is our recommendation that BNM develop its technology in conjunction with

these new methods in order to capitalize on this trend. As BNMs platform will

theoretically require less fetal DNA, it is well suited to fill in this emerging market

need. Listed below, in Table 2, are several companies in this emerging field which

BNM could potentially collaborate with.

Table 2. Selected companies developing noninvasive prenatal

genetic diagnostics methods. (Adapted from IN VIVO, Vol. 27, No. 10, Nov. 2009).

Company Genetic

material/Source

Comments

Sequenom Cell free nucleic acid

from blood

Developing down syndrome test

using mass spec and sequencing-based detection technology.

Artemis Health Cell free nucleic acids /

Whole fetal cells from

blood

Initially developing whole fetal cell

separation methods using

microfluidics. In January 2009

added a second program to develop

technology for sequencing-based

detection of fetal nucleic acids for

down syndrome.

Lenetix Cell-free nucleic acidsfrom blood

In clinical trials of its PloidYXarray based technology using DNA

methylation differences to detect

trisomies.

Ravgen Cell free nucleic acids

from blood

Treat blood samples with

formaldehyde to boost proportion

of fetal DNA in the samples.

Published results in Lancet

Zoragen Cell free nucleic acids Detection of unpaired nucleic acidsfor prenatal diagnostics

Celuda Fetal cells from blood Isolation of cells partly based on

cell sorting technology in licensed

from Genoptix.


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Ikonisys Fetal cells from

cervical smear

Digital microscopy imaging

technology-using FISH probes on

fetal cells isolated from cervical

smear.

Parsortix Fetal Cells from blood Device to separate fetal cells from

1.5 ml of whole blood

SCIENTIFIC EXPLORATION

Within the context of the prenatal genetic testing market, the BNM core technology

in its present state is most suited to detecting gross chromosomal aberrations

including deletions, duplications, and translocations. The following section presents

an overview of high-prevalence chromosomal abnormalities and the diseases

associated with them, where BNM technology can potentially be applied.

The core BNM protocol involves an initial nickase labelling step that generates a

signal which is then read by NanoAnalyzer device. Specifically, Sample DNA is

nicked by enzymes (nickases) in a sequence-specific manner, and these nicks are

then repaired by incorporating fluorescent nucleotides which can be detected by the

NanoAnalyzer. In its current form the technology platform achieves a resolution of

2.5 kilobases between motifs, and can process 10 genomes in 30 minutes. The nick-

labeling scheme provides a very specific readout due to the requirement of two

enzymatic reactions (DNA nicking by nickase, and fluorescent nucleotide

incorporation by polymerase) for a signal output.

Overview of chromosomal abnormalities

Down Syndrome

Down syndrome is a chromosomal abnormality characterized by the presence of an

extra copy of genetic material on the 21st chromosome (Trisomy 21). Trisomy 21 is

usually caused by nondisjunction in the gametes prior to conception, and all cells in

the body are affected. However, when some of the cells in the body are normal and

other cells have trisomy 21, it is called mosaic Down syndrome. The effects of the

extra copy vary greatly among people, depending on the extent of the extra copy,

genetic history, and pure chance. The incidence of Down syndrome is estimated at

one per 800 to one per 1000 births. In 2006, the Centers for Disease Control and

Prevention estimated the rate as one per 733 live births in the United States (5429

new cases per year). Approximately 95% of these are trisomy 21. Down syndrome

occurs in all ethnic groups and among all economic classes.


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Sex chromosome aneuploidies

Chromosome 23 is susceptible to a variety of aneuploidies resulting from

nondisjunction during meiosis or mitosis. The most common sex chromosome

aneuploidies are Turner's Syndrome (XO), Kleinfleter's (XXY), XYY, and XXX with a

prevalence of ~1/1000 live births, comparable to Down's Syndrome. Pre-natal

screening for sex chromosome aneuploidies is identical to Down's Syndrome (i.e.fetal cell extraction via amniocentesis or CVS followed by karyotyping or FISH).

Neural tube defects (NTDs)

Neural Tube Defects (NTDs) are caused by the neural tube not fully closing in

development. The neural tube forms 28 days after conception, so this is the earliest

that this problem can be identified. There are many different manifestations,

depending on the manner in which the tube does not close. One out of every 1000

babies have a neural tube defect. However, many manifestations of NTDs cause the

baby to be stillborn. For babies that are born alive, they are critically disabled, and

almost always unable to live a normal life. The tests for neural tube defects are

identical to those used for prenatal genetic testing (maternal serum alpha

feroprotein testing and ultrasound, follow by amniocentesis if it is determined there

is a high probably of a defect). NTDs are caused by a mixture of environmental and

genetic factors. High folic acid intake by a mother can decrease the likeliness of a

defect, while maternal insulin dependent diabetes and the use of anti-seizure

medication increases the chances of a defect. The genetic impact can be seen

because normally there is only a 0.1% chance of a defect in a first child, but the

chances increase to 2-5% if a couple's first child had a defect. NTDs are common

affects of Trisomy 13, Trisomy 18, Mecket-Groubert Syndrome, and more. Duke

University is currently doing SNP research, genome wide, to determine which genes

are involved in NTDs. Duke University currently doing SNP research, genome wide,

to determine which genes are involved.

Cardiac chromosomal abnormalities

The congenital cardiac diseases include congenital restrictive cardiomyopathy,

hypertrophic cardiomyopathy, dilated cardiomyopathy. If the disease has

manifested sufficiently in a patient, it can be screened for with a thorough physical

exam or an echocardiogram, which costs approximately $130 for a hospital to

perform, and diagnosed with echocardiography. Long QT syndrome may also be

congenital, in which case it is a congenital cardiac channelopathy. All of these

diseases are very rare, and represented by a wide range of genetic mutations,predominantly point mutations. Additionally, these diseases have variable

penetrance so that some people may have one of these diseases but never have

clinical manifestations, while other individuals may be more severely affected.

Congenital restrictive cardiomyopathy is by far the least common of these diseases.

The disease itself represents an inability of the heart to stretch out during its

relaxation phase (diastole). It is diagnosed by echocardiogram.


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Congenital hypertrophic cardiomyopathy is a disease in which the ventricular walls

are thickened, leading to mitral prolapase, poor cardiac output, and potentially fatal

arrhythmias. This is the most common cause of sudden death of young athletes who

die during an athletic event (e.g. young basketball star who suddenly dies in the

middle of a game). A physician may first suspect the disease during the physical

exam, and may confirm the diagnosis with an ECG and echocardiogram. There are

individual point mutations which can determine whether the disease phenotype is

mild or carries a high chance of sudden death. Early diagnosis allows physicians to

recommend that the affected individual avoid potentially dangerous activities. A list

of the known genetic mutations can be found at (11).

Congenital dilated cardiomyopathy is a disease in which all four chambers of the

heart are expanded and have thin walls. This may also be related to neuromuscular

disorders, mitochondrial disorders, carnitine deficiency, Barth syndrome, or Naxos

disease. This is normally diagnosed by ultrasound after detected on physical exam

by a physician.

Long QT syndrome is an electrical conduction disorder of the heart which makes

individuals susceptible to Torsade de Pointes when placed on certain drugs. This is

generally diagnosed by ECG.

Deletions

The most common deletions are DiGeorge (22q), Smith-Magenis (17p), Wolf-

Hirschorn (4p), Angelmann (15q), Prader-Willi (15q), Williams (7q), Miller-Dieker

(17p), Cri-du-chat (5p), Rubenstein-Taybi (16p or 16q). Generally these deletions

manifest early on in child development as cognitive delays and in some specific

instances can be correlated with e.g. cardiac abnormalities as in the case of

DiGeorge's. Currently, these deletions are not screened for pre-natally as their

prevalence is low. With the exception of DiGeorge (~1/4000 live births) the deletions

are in the < 1/20,000 prevalence range.

Potential application areas

The tables below list the diseases (classified based on the size of structural

aberration) and their corresponding genetic abnormality, prevalence and the

potential of BNMs technology to diagnose the condition.

To show proof of principle, only the recognition sequence of nickase BtsI1 was tested.

As seen from the following tables, the technology in its current form, is sufficient to

diagnose diseases arising from chromosomal duplications to small structural

abnormality at the gene level.


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Table 3. Disorders associated with aneuploidy

Disease Abnormality Prevalence

Down syndrome Trisomy 21 1 in 800

Klinefelter's syndrome XXY 1 in 1000

XYY syndrome XYY 1 in 1000

Triple X syndrome XXX 1 in 1000

Turner syndrome XO 1 in 2500

Edwards syndrome Trisomy 18 1 in 3000

Patau syndrome Trisomy 13 1 in 25000

Tetrasomy XXXX, XXXY Extremely rare

Table 4. Disorders associated with large chromosomal deletions

Diseases Abnormality Prevalence

DiGeorge syndrome 22q11.2 1 in 4000

Williams syndrome 7q11.23 1 in 10000

Prader-Willi syndrome 15q11-13 1 in 15000

Smith Magenis syndrome 17p11.2 1 in 25000

Cri du chat syndrome 5p15.2 1 in 30000

Wolf-Hirschhorn syndrome 4p16.3 1 in 50000

Rubenstein-Taybi

syndrome 16q13 1 in 125000

Langer-Giedion syndrome 8q23.2 to 8q24.1 Extremely rare


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Table 5. Disorders associated with gene deletions

Diseases Abnormality Prevalence

Fragile X syndrome FMR1 gene (Xq28) 1 in 5000

Angelman syndrome UBE3A gene (15q11-13) 1 in 15000

Alagille syndrome JAG1 gene (20p12.2) 1 in 100000

Miller-Dieker

syndrome LIS1 gene (17p13.3) Extremely rare

Diseases and their corresponding genetic loci

1. Down syndrome Trisomy 21

Also known as Trisomy 21, a condition in which one has an extra 21 chromosome .

2. Klinefelter's syndrome XXY

A chromosomal abnormality in males due to the presence of an extra X chromosome.

3. XYY syndrome XYY

It is an aneuploidy in which males have an extra Y chromosome.

4. Triple X syndrome XXX

Also known as Trisomy X, a condition in which females have an extra Xchromosome.

5. Turner syndrome XO

Turner syndrome results from the absence of part or all of the X chromosome.


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6. Edwards syndrome Trisomy 18

Also known as Trisomy 18, a condition in which one has an extra chromosome 18.

7. Patau syndrome Trisomy 13

Also known as Trisomy 13, a condition in which one has an extra chromosome 13.

8. Tetrasomy XXXX, XXXY

Aneuploidies arising from tetrasomy in sex chromosomes.

9. DiGeorge Syndrome Deletion 22q11.2

A genetic disorder that arises from the deletion of q11.2 region in chromosome 22,

resulting in congenital heart disease and neuromuscular problems.

10. Williams Syndrome

Deletion 7q11.23

Williams syndrome is a neuro-developmental disorder caused by the deletion of

q11.23 in the long arm on chromosome 7.

11. Prader-Willi syndrome

Deletion 15q11-13


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Prader-Willi syndrome is a very rare chromosomal disorder that causes several

growth defects such as low muscle tone, short stature and mild mental retardation.

It is often misdiagnosed as Down syndrome.

12. Smith Magenis Syndrome

Deletion 17p11.2

Smith-Magenis Syndrome (SMS) is a genetic disorder that impairs development

resulting in mental retardation, sleep disturbances and distinctive facial features.

13. Cri du chat

Loss of a small region in band 5p15.2

Cri du chat syndrome arises from the deletion of region p15.2 in chromosome 5 that

results in impaired cerebral development.

14. Wolf-Hirschhorn

Deletion 4p16.3

Wolf-Hirschorn syndrome is caused by the deletion of region p16.3 in chromosome 4

resulting in mental retardation, microcephaly and poor muscle tone.


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15. Rubinstein-Taybi Syndrome

Deletion 16p13

Rubinstein-Taybi Syndrome is a genetic disorder that results in short stature,

learning difficulties and skeletal abnormalities.

16. Langer-Giedion syndrome

Deletion 8q23.2 to q24.1

Occurs from the loss of region q23.2-24.1 in chromosome 8 and results in learning

disabilities and impaired skeletal development.

17. Fragile X syndrome

FMR1 gene (Xq28)

Fragile X syndrome is a genetic disorder arising from the expansion of single

trinucleotide (CGG) sequence on the X chromosome that results in a failure to

express the FMR1 gene required for normal neural development.

18. Angelman syndrome

UBE3A gene (15q11-13)

A genetic disorder that arises from the deletion of q11-13 region of chromosome 15

that causes an absence in UBE3A gene expression required for normal brain

development.


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19. Alagille syndrome

JAG1 gene (20p12.2)

A genetic disorder that arises from the microdeletion of 20p12 chromosome

corresponding to the JAG1 gene required for normal embryonic development.

20. Miller-Dieker syndrome

Deletion of part of 17p (which includes both the LIS1 and 14-3-3 epsilon gene).

Miller-Dieker syndrome is a developmental defect caused by incomplete neuronal

migration.

Detailed information for the above mentioned 20 diseases are available through the

following references. The reference numbers are matched up to the disease numbers.

1. http://emedicine.medscape.com/article/943216-overview2. http://emedicine.medscape.com/article/945649-overview3. http://ghr.nlm.nih.gov/condition=47xyysyndrome4. http://ghr.nlm.nih.gov/condition=triplexsyndrome5. http://emedicine.medscape.com/article/949681-overview6. http://emedicine.medscape.com/article/943463-overview7. http://emedicine.medscape.com/article/947706-overview8. http://en.wikipedia.org/wiki/Tetrasomy9. http://emedicine.medscape.com/article/886526-overview10. http://emedicine.medscape.com/article/893149-overview


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11. http://emedicine.medscape.com/article/947954-overview12. http://ghr.nlm.nih.gov/condition=smithmagenissyndrome13. http://emedicine.medscape.com/article/942897-overview14. http://emedicine.medscape.com/article/950480-overview15. http://emedicine.medscape.com/article/948453-overview16. http://ghr.nlm.nih.gov/condition=langergiedionsyndrome17. http://emedicine.medscape.com/article/943776-overview18. http://ghr.nlm.nih.gov/condition=angelmansyndrome

19. http://ghr.nlm.nih.gov/condition=alagillesyndrome20. http://ghr.nlm.nih.gov/condition=millerdiekersyndrome

INTELLECTUAL PROPERTY

Potential issuesThere are three potential IP issues that need to be examined. They are the method

being used, the materials being used, and the particular DNA sequences being

identified. The first two issues are not of particular concern. The method being

used is a unique process created by Bionanomatrix. The materials being used, such

as nickases, are also not of concern. While these materials potentially are patented,

as they are purchased this satisfies the patent concerns.

The primary IP issue pertains to the specific DNA sequences and aberrations being

detected and identified. DNA sequences can be patented. The rationale behind this

is that the normal method of patenting methods is not sufficient with DNA. This is

because there are numerous methods of detecting the presence of genes. Suppose if

patents for DNA sequences did not exist, and in this setting, Company A identifiesthe link between a gene and a disease. If Company A is only able to patent their

method for detecting for the presence of the gene, Company B could develop a new

method, and take control of the market. The idea behind allowing for DNA

sequences to be patented is that it will provide incentive to discover the links

between genetics and diseases.

However, this incentive comes at a price as it discourages research into additional

effects of patented genes. To combat this reaction, royalties associated with DNA

sequence patents are kept low. This therefore provides incentive to initial research,

while not making future research prohibitively expensive.

The genetic diseases being screened for all involve specific DNA sequences that have

been linked to a disease. Therefore, it is likely that all of these sequences have been

patented. However, as is evidenced by the fact that there is currently a large

market for these tests, the royalty cost is not prohibitively expensive. There are two

possible explanations for this. First, for genetic links that have been known for a

while, the patents may have expired. This is likely true for diseases which the


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genetic markers have been know for a long period of time, such as is true for Downs

Syndrome. Second, for genetic links that have been discovered more recently and

are currently tested for, the royalties must be reasonable.

There are two potential areas of concern. One is diseases that are not currently

tested for that are still patented. As Bionanomatrixs technology will be able to test

for a much larger number of diseases than previous tests, it is possible that some of

these new sequences have high royalties. However, this is unlikely because of the

practice of keeping royalties on DNA sequences low. The second potential problem

is adding up royalties. Although Bionanomatrix may be able to test for an unlimited

number of diseases at one time, if too many are still patented, this cost may add up.

This may provide a limit as to the number of diseases able to be tested for at one

time. However, as SNP Chip and CGH test for a similarly large number of diseases,

it is unlikely this will be a major problem. For an additional analysis for this

problem, please consult (12).

In conclusion, although there are potential IP obstacles, they are the same problemscompetitors currently in the market face. If Bionanomatrix is going to be testing for

the same diseases as its competitors do, then patents of DNA sequences will not be

an issue. If Bionanomatrix is planning on testing for different DNA sequences, then

it will be necessary to determine if that sequence is covered by a patent, and the cost

of the royalty if a patent does exist. If a patent does exist, it will then have to be

determined in light of the royalty if it is still profitable to test for this additional

DNA sequence.


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MARKETANALYSIS

Global market for molecular diagnostics

The most common assays in the overall molecular diagnostics market are those for

genetic/chromosomal tests, including various cancer-related tests, karyotyping,

genetic disease carrier testing, prenatal diagnosis, sex determination, and

susceptibility testing. This currently accounts for about 60% of all molecular

diagnostic testing. The remainder of the total molecular diagnostics arena includes

infectious disease testing with 36.3%, and other applications like identity/blood bank

screening.

Table 6. Distribution of molecular diagnostics testing market in

2009 (Source BCC).

Application Percent

Infectious disease diagnostics 36.3

Oncology diagnostics 31.8

Prenatal and newborn screening 25.6

Identity/Blood bank/other 6.3


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Global gene/chromosome diagnostics market

In 2008, the global gene chromosome (DNA) diagnostics market was valued at

approximately $10.6 billion. The total market is projected to grow at a compound

annual growth rate (CAGR) of 13.7% to reach $20.1 billion by 2013. Within the

global DNA diagnostics market, the market for PCR-based diagnostic assays claimed

the largest share in 2008 amounting to an estimated $5.39 billion. The PCR

diagnostics market is projected to grow at a compound annual growth rate (CAGR)

of 11.9% to reach nearly $9.5 billion by 2013. The market for microarray diagnostic

assays was valued at $2.4 billion in 2008 and is projected to grow at a compound

annual growth rate (CAGR) of 13.8% to reach $4.6 billion by 2013. The market for in

situ hybridization diagnostic assays was valued at $1.9 billion in 2008, and is

projected to increase at a compound annual growth rate (CAGR) of 15.3% to reach

$3.9 billion by 2013.

Table 7. Global market for gene/chromosome diagnostics by

segment ($ Millions, Source BCC)

PCR-based assays 5,389

DNA microarrays 2,396

FISH diagnostics 1,890

Other 945

Global market for DNA microarrays

The global market for DNA Microarray or clinical diagnostics was $2.4 billion in

2009 and estimated to grow at a rate of 13.8%. The global biochip market by end

use is shown in the table below (Table 8). Drug discovery and development and

diagnostics are large consumers of biochips. Whole genome association studies and

next-generation DNA sequencing market segments are important drivers of future

growth in these markets. Diagnostics applications for biochips are rapidly

expanding, and this market segment is expected to experience high future growth.

By 2014, drug discovery and development, and diagnostics applications are expected

to represent more than 71% of the overall biochip market, emphasizing the future

importance of these devices to the medical and pharmaceutical industries.


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Table 9. Market distribution for molecular diagnostics for

inherited genetic disease testing in the US, ($ millions)

Prenatal 240

Newborn 53.7

Hetrozygote 15.8

Presymptomatic 6.32

Total 315.8

Table 10. Market driving forces

Market Driving Force Significance for Biochip Demand

Growth in life science R&D

funding

Drives demand for biochips in research tools, drug discovery

and development

Growth in large-scale

biochip, genomic and

proteomic initiatives

Standardizes biochip products across platforms and provides high visibility

Testing sites for biochip products

Need for more efficient

drug discovery and

development

Creates incentive for novel biochips technologies that provide high throughput

and multiplex capability at lower prices

Development of

personalized medicines

Creates strong demand for pharmacogenetic (PGx) tests that use biochips

Need for early cancer

detection

Drives biomarker discovery and diagnostic applications for biochips

Aging populations in U.S.,

Europe, and Japan

Increases demand for MDx tests in cancer and other diseases

New discoveries in

genomics and proteomics

Increases growth opportunities in novel biochip products

Disposability, small

sample sizes

Drives growth for miniaturized biochips in diagnostics market segment

Rapid decline in genetic

analysis costs

Enables widespread adoption of biochips by research and drug discovery markets


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Discovery of more gene

and/or SNP- disease

associations

Drives demand for biochip consumables and instruments in research and

diagnostics applications

Competitive Analysis

Currently the standard technologies used for pre-natal diagnostics are karyotyping

and FISH. For post-natal screening, the current dominant technologies are array-

CGH and SNP chips. To determine the practical details of the currently employed

technologies as well as to gain insights into emerging market trends, we have

conducted extensive literature and primary surveys of technicians and clinicians at

CHOP (Childrens Hospital of Philadelphia), HUP (Hosptial of the University of

Pennsylvania), Johns Hopkins Hospital. The results are discussed below and

summarized in the Table 11.

Cost per use (reagents, labor/technical staff requirements, time

additional per run)

The cost of the molecular markers used in FISH is ~ $20 - $30 per marker [primary

survey of HUP clinician]. Hybridization requires overnight incubation of the cells

with the molecular markers and quantification of the results is done manually, with

the goal of observing ~ 20 labelled cells out of ~200 cells in a typical amniotic fluid

extraction sample. The cost of Illumina's 610 beadchip analysis is $400/ sample, as

determined via a consensus survey of online academic sequencing centers. The costs

of other comparable chip-based platforms such as those of Affymetrix and Agilent

can also be found at (13). The total time for a run on the Illumina platform is ~ 6

days with a breakdown of 1-2 days to isolate DNA from blood and 3-4 days of

runtime on the beadchip [primary survey of CHOP technician].

Advantages, limitations, efforts to improve that currently used

technology

Whereas the accuracy of karyotyping is quite high (> 99%) a fact which has no doubt

contributed to its longevity as the "gold standard" in cytogenetic screening of large

scale chromosomal aberrations, the accuracy of FISH is relatively poorer (~ 70%)(13). This is due in large part to human error, as nearly overlapping fluorescent

spots must be discriminated manually under a microscope in typical FISH protocols

[primary survey]. This relatively large error rate could be the basis of a competitive

advantage for BNM's technology platform over FISH.

FISH is a highly accurate test for detecting the standard 22q11.2 deletion when a

couple has had a previous child with the deletion or a parent has the deletion.


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However, DiGeorges (DGS) is a heterogeneous disorder and 1015% of individuals

with the characteristic features do not have the standard 22q11.2 deletion (14) .

These atypical deletions of 22q11 have not been detected by FISH using

commercially available probes. Therefore, FISH has an estimated false negative rate

of 10-15% for DGS. For a pre-implantation genetic screen (PGS) done prior to IVF,

it was found that some abnormalities could not be detected by the seven-probe panel

(13, 16, 18, 21, 22, X and Y) used in fluorescence in-situ hybridization that were

detected with CGH. This data was generated from patients with reoccurring IVF

failure due to aneuploidy. FISH has been used with probes for up to 15

chromosomes.

The primary advantage of BNM technology over karyotyping is speed. Karyotyping

requires cells to be in metaphase, and this fact requires the amniotic sample to be

cultured for 1 week in order to achieve a sufficient yield of ~ 20-50 metaphasic cells

for fixing and displaying analysis. In principle, BNM technology which requires only

~ 10-100 cells worth of DNA, would obviate the need for culturing. As an indicator

of current trends in post-natal genetic screening, Children's Hospital of Philadelphia

(CHOP) uses SNP chips almost exclusively to screen for developmental disorders in

infants [primary research]. They perform the testing in-house and utilize the

Illumina 610 quad bead chip. The Illumina 610 quad beadchip is capable of

producing genotype calls in over 590,000 SNPs, an average genomic marker spacing

of 1 SNP per 6 kbp. The average call rate, the proportion of all SNPs called across

each sample, for Illumina's 610 quad beadchip platform is > 99% [primary survey of

CHOP technician and (15)]. The false positive/false negative rates are dependent on

the specific characteristics of the genetic disorder and are therefore not quoted as an

intrinsic spec in the platform's technical performance literature. However,

interviews with a CHOP technician indicated that accuracy is not an issue of

concern in analyses carried out on the Illumina platform.

The potential advantages of BNM's platform over chip-based technologies such as

Illumina 610 platform are speed, and sensitivity to certain types of mutations such

as balanced translocations and insertions of tandem repeats. Illumina 610 requires

3-4 days of runtime per sample [primary survey CHOP technician], whereas BNM is

potentially shorter. Detection of balanced translocations and tandem repeats is not

straightforward using SNP, FISH, or array-CGH, whereas it is an intrinsic strength

of BNM's platform.

For the Illumina 610 platform used at CHOP, the analysis is done on DNA isolated

from blood. Typically, 1-2 mL of blood is extracted, yielding ~ 1 million white blood

cells from which DNA is extracted using Qiagen's PureGene kit. This step takes

about a day of technician labor [primary survey of CHOP technician].

Typically, array-CGH procedures use between 50,000 to 500,000 cells of specimen

DNA in the labeling reaction (16) . If BMN's application can perform analysis on


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smaller samples, than it will have a sizable advantage over array CGH applications,

which are often limited by the amount of specimen DNA. This advantage can be

realized in a faster turnaround time and lower cost, as other competing applications

require cell culturing and expansion when the sample size too small.

BNM's application may also be better equipped to detect and quantify copy-number

changes than array CGH platforms. It has been found for array CGH that the

signal intensity element is affected by a number of factors, including base

composition, proportion of repetitive sequence content and amount of hybridizable

DNA in the array element (16) . This has resulted in intensities varying by a factor

of 30 or more with no change in copy-number. This feature may create opportunity

for BNM's technology to fulfill unmet detection needs in diseases copy number

alterations.

Table 11. Summary Table of Competitive Technologies

Sources:

a: Primary Research

b: http://www.solexa.co.uk/downloads/Illumina2008ProductGuideUpdate.pdf

c: http://www.broadinstitute.org/gen_analysis/genotyping/pricing.html

Risks of addressing this market at this time.

Sample Requirements Time

Requirements

Cost Breakdown Consumer

Cost

Accuracy

type amount Total

time

(days)

hands

on

(hours)

Equipment Reagents Labor

Karyotype Fetal

cells

20-50 cellsa

(metaphase)

7 -10a

6a

Standard ~$200 $700a

~ 99%a

FISH Fetal

cells

20-50 cellsa

(interphase)

1-2a

6a

Standard $30/probea

~$200 $1000 [5

diseases]a

~ 70%

(13)

ArrayCGH Blood

(WBC)

50,000-

500,000

cells (16)

3-4 4 Standard,

Reader,

chip

Label Kit ~$120

SNP Blood

(WBC)

200 ng

DNAb

3-4a

4a,b

Standard,

Reader,

chip

Label Kit ~$120 $372c > 99%

a.b

(15)


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Using the microarray technology market as an indicator, there has been slower-

than-expected growth with new applications using unconventional approaches. This

market resistance to new technologies may be a barrier to BNM technology. It is

expected that the research markets will grow much faster than clinical applications

(17).


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CANCER GENETIC TESTING

BackgroundCancer genetic testing represents a potentially significant market opportunity for

Bionanomatrix technology. The various types of cancers are collectively of enormous

public health significance. In 2002, approximately 1.3 million new cases of cancer

were diagnosed in the United States, and about 0.6 million deaths were attributed to

cancer, making it the second leading cause of death (18).

Currently, genetic tests are used in the diagnosis and management of only a small

number of cancers. This is primarily due to the fact that the molecular mechanisms

of most cancers are not known, or do not cluster consistently into a small number of

categories, and due to the difficulty in obtaining high-resolution genetic informationfrom patient DNA. However, progress in both areas is continually increasing the

scope of genetic testing in the diagnosis and management of cancer, as is already the

case in some cancers such as leukemia, where genetic testing constitutes an integral

part of the treatment and management program (19).

Patients for whom cancer genetic testing is performed can be roughly divided into

two main categories: patients actively manifesting symptoms, and patients not

manifesting symptoms but considered to be at risk due to family history. Genetic

tests based on Bionanomatrix technology can potentially cater to both groups.

All cancersSince all cancerous cells share some general features, certain genetic tests can be of

use in all cancers. Aneuploidy in cells (i.e. cells having an abnormal number of

chromosomes) is often indicative of cancer, and is almost always observed in all

cancers (20). Furthermore, in some cancers such as ovarian cancer, there is a strong

association between the extent of aneuploidy and the progress of the disease, and

aneuploidy tests are an integral component of the information considered to assess a

patients prognosis, and are also being used in therapy planning (21). Since fast,

inexpensive, and sensitive DNA aneuploidy tests based on staining and flow

cytometry already exist, it would be hard for Bionanomatrix technology to compete

in this space, unless it is offered in a basket of multiple tests.

Leukemia

Leukemia is the term used for a group of cancers of the blood and bone marrow

which are typically characterized by abnormally high levels of leukocyte


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proliferation. In 2002 there were approximately 31,000 new cases of leukemia, and

22,000 deaths attributed to leukemia, in the United States.

Acute myeloid leukemia, or AML, is a leukemia subtype in which abnormal

proliferation of leukocytes in the bone marrow hinders hematopoiesis, the

production of normal blood cells; in 2002, approximately 11,000 new cases of AML

were diagnosed, and approximately 7,000 deaths were attributed to AML. AML is

perhaps one of the best studied cancers, and genetic lesions associated with AML

usually involve large-scale translocations or inversions. Genetic testing plays an

integral role in the contemporary management of AML; the National Comprehensive

Cancer Network considers cytogenetics to be the single most important prognostic

factor for predicting remission rate, relapse, and overall survival. (19)

Currently, genetic testing is being used to diagnose AML, to classify patients into

AML subtypes for purposes of targeted treatment, and assess prognosis (survival

and relapse rates can vary dramatically depending on subtype), to predict how a

patient with a particular genetic lesion will respond to a specific treatment, and alsoto monitor the status of the disease (active versus latent). These tests have for some

time been performed with genome scale low-resolution assays such as karyotyping

and FISH. However, since approximately half of adult AML patients have a normal

karyotype, high-resolution assays such as RT-PCR, expression profiling via

microarrays, and sequencing are becoming increasingly popular.

A recent review (22) highlights the added benefits of obtaining high-resolution

genetic information from AML patients, and outlines a protocol in which

increasingly higher resolution assays are used to place patients into specific disease

subtypes; the review also states the future prospect of monitoring the state of the

disease at a very high resolution using arrays, sequencing, etc. Since prohibitively

high costs and sample requirements (number of cells, extraction, etc) are considered

to be the primary barriers to adoption of high-resolution assays, this can be a

potential market opportunity for Bionanomatrix technology, especially if it can be

used to perform these analyses at significantly lower costs than competing

technologies such as SNP arrays.

Colorectal cancer

Colorectal cancers constitute the third-leading cause of cancer-related deaths in the

United States; in 2002, approximately 150,000 patients were diagnosed, and 50,000

deaths were attributed to colorectal cancers (18). Patients diagnosed with colorectal

cancer generally fall into two categories, those with strong family history of

colorectal cancer comprise about 25% of the population, and the rest whose disease

is considered to be sporadic.

Colorectal cancer generally begins with the inactivation of the APC tumor-

suppressor gene (adenomatous polyposis coli, which has been detected to be mutated


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in 75% of sporadic colorectal cancers) as the initial neoplastic event, and progresses

further with subsequent mutations in the oncogene KRAS, and tumor suppressor

genes DCC and TP53.

The genomic instability associated with colorectal cancer can be divided into two

categories: microsatellite instability, which is most commonly associated with

hereditary colorectal cancer, and chromosomal instability, which is most commonly

associated with sporadic colorectal cancer. Microsatellites are repetitive sequences

of DNA spread throughout the genome. Microsatellites can cause additions or

deletions to be made during the copying process because the DNA polymerase can

slip on repetitive sequences. These errors are usually corrected by the proteins

involved in the mismatch repair system, but can accumulate and cause mutations in

coding and non-coding regions if mismatch repair activity is reduced. Inactivation of

one or more of the following genes has been observed to cause microsatellite

instability in colorectal cancer: MLH1, MSH2, PMS1, PMS2, MSH6, EXO1 and

MLH3 with the majority of sporadic colorectal tumors with microsatellite instability

showing mutations in MLH1 (90%) or MSH2 (5%). Chromosome instability refers

to the loss or gain of whole chromosomes (aneuploidy), or the loss of one of the

parental alleles (loss of heterozygosity or LOH).

Bionanomatrix has potential market opportunity in both familial and sporadic

colorectal cancers. Individuals carrying a mutation in one of the aforementioned

mismatch repair genes have a lifetime risk of developing colorectal cancer that is as

high as 80%. Genetic tests usually involve the use of microsatellite instability

analysis, and sequencing of specific regions to screen for mutations in mismatch

repair genes. However, since these tests are expensive and time-consuming, they

are offered only to candidates considered to be at high risk. Furthermore, this panelof genetic tests (detailed in (23)) fails to identify a significant fraction of genetic

lesions that may eventually cause colorectal cancer in patients. The ability to

interrogate the patients entire genome for microsatellite instability in a cost-

effective manner can significantly improve the quality of care.

A market opportunity exists if Bionanomatrix technology can be utilized to offer

existing genetic tests at significantly lower costs. Furthermore, if Bionanomatrix

technology can be harnessed to detect microsatellite instability throughout the

patients full genome, it can potentially improve the quality of care offered to

patients. Microsatellite instability is not specific to colorectal cancer, and is

generally considered a strong indicator of genomic instability, which in turn is a

reliable predictor of neoplastic progression. Hence, fast and inexpensive

microsatellite instability assays could be commercially viable in a number of

different categories.


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Table 12. Currently available cancer genetic tests.

The following table contains information from a cancer test database published in a

report commissioned by the Department of Health and Human Services. Onlygenetic tests for which Bionanomatrix technology in its current form can be

employed are listed. This filters out the majority of entries since most cancer tests

involve antibodies to detect specific molecular markers, and also since a large

number of genetic tests involve sequence-level resolution.

Name Other Names

Primary

Secondary

Prevention

Diagnos

tic

Pro

nostic

Recurrence

Monitoring

Specimen

AML1/ETO translocation t(8;21) x x x x blood,marrow

B-cell gene

rearrangementx x x blood,

marrow,

tissue

BCL-1/JH gene

rearrangement

t(11;14) x x x blood,marrow,

tissue

BCL-2 translocation t(14;18) x x x x blood,marrow,

tissue

BCR/ABL gene

rearrangement

Philadelphia chromosome x x x x blood,marrow

BRCA Analysis BRCA1, BRCA2 x x blood

Chromosome 18q assay 18q/RER, DCC x x Blood, tissue

Colaris MLH1, MSH2x x

Blood

Colaris AP APC, FAP x x Blood

FLT 3 mutation x Blood

HER-2/neu c-erbB-2, PathVysion,

Herceptin eligibilityx x Tissue


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IgVH mutation analysis x Blood,marrow

Microsatellite instability MSI, BAT-26, RER+, HNPCC x x x x Blood, tissue

MLH1, MSH2, MSH6

mutations

HNPCC mismatch repair gene x Blood

Oncotype Dx Breast cancer assay x Tissue

p53 tumor suppressor

gene

p53 x Tissue

PML/RARA translocation t(15;17) x x x Blood,marrow

PreGen-26 MSI, BAT-26 x x x Stool

PreGen-Plus k-ras, APC, p53 x x Stool

T-cell receptor gene

rearrangementx Blood,

marrow,

tissue

TEL/AML1 gene fusion t(12;21) x x x Blood,marrow

Genetic Tests for Cancer. Technology Assessment. January 2006. Rockville, MD: Agency for Healthcare

Research and Quality. http://www.ahrq.gov/clinic/ta/gentests/.


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