Exhibit 10.5

FIRST ADDENDUM

TO THE RESEARCH AND LICENSE AGREEMENT

This First Addendum to Research and License Agreement (the “First Addendum”) is
made by and between The Technion Research & Development Foundation Ltd. (“TRDF”)
and Eloxx Pharmaceuticals Ltd. (“Licensee” or “Eloxx”).

WHEREAS, TRDF and Eloxx are parties to Research and License Agreement with an
effective date of August 29th, 2013, as amended on November 26, 2013,
January 14, 2014 and June 9, 2014 (the “Agreement”); and

WHEREAS, the parties desire to continue the relationship contemplated by the
Agreement and therefore to further amend the Agreement as set forth herein;

NOW, THEREFORE, the parties hereby agree as follows:

 

1. Unless otherwise defined herein, capitalized terms used in this First
Addendum shall have the meanings assigned thereto in the Agreement.

 

2. The Parties wish to add new specific research work under this First Addendum,
all as described in Exhibit D1 attached hereto which shall be added to Exhibit D
of the Agreement (the “First Addendum Research”).

 

3. For the First Addendum Research, a separated budget is required to be paid by
Eloxx to TRDF, all as agreed upon and set in Exhibit D1, in the total amount of
thirty thousand US dollars ($30,000) (the “First Addendum Budget”).

 

  3.1 Licensee shall fund the First Addendum Budget to be performed within 3
months commencing as of the execution of this First Addendum, as follows:

 

  3.1.1 First installment of ten thousand US dollars ($10,000) shall be paid
upon the execution of this First Addendum.

 

  3.1.2 Second installment of ten thousand US dollars ($10,000) shall be paid no
later than June 30th, 2014.

 

  3.1.3 Third installment of ten thousand US dollars ($10,000) shall be paid
upon completion of the First Addendum Research and supply to Eloxx of related
materials.

 

  3.2 VAT as applicable on time of payment, shall be added to each installment.

 

  3.3 TRDF shall issue a proper invoice for each installment.

 

4. Except as added herein, all Confidential terms and conditions of the
Agreement shall remain in full force and effect, as relevant to the First
Addendum Research.

 

1

--------------------------------------------------------------------------------

5. This First Addendum may be executed in counterparts, each of which shall be
deemed an original and all of which together shall constitute one and the same
instrument. Any signature page delivered by facsimile or electronic image
transmission shall be binding to the same extent as an original signature page.

IN WITNESS WHEREOF, the parties hereby accept and agree to the terms and
conditions of this First Addendum.

 

ELOXX PHARMACEUTICALS LTD.  

THE TECHNION RESEARCH & DEVELOPMENT FOUNDATION LTD.

  By:  

/s/ Silvia Noiman

    By:  

/s/ Benjamin Soffer

  Name: Silvia Noiman     Name: Benjamin Soffer   Title: CEO     Title:
Technology Transfer Office, Manager   Date: 3/8/14     Date: 07.14.2014  

--------------------------------------------------------------------------------

Exhibit D-1

--------------------------------------------------------------------------------

Submitted to Eloxx Pharmaceuticals LTD

Research Plan

Title

Development of Aminoglycose-Based Drug for Treatment of Human Genetic Diseases
and Many Forms of Cancer Caused by Nonsense Mutations

I. Scientific and Technological Background

Nonsense mutations are in-frame premature termination codons (PTCs) that convert
a sense codon of mRNA to UAA, UAG or UGA stop codon and lead to the production
of truncated, nonfunctional proteins. PTCs are responsible for more than 1,800
inherited human diseases, including cystic fibrosis (CF), Duchenne muscular
dystrophy (DMD), Usher syndrome (USH), Hurler syndrome (HS) and numerous types
of cancer. For many of those diseases there is presently no effective treatment
and the only treatment widely used is symptomatic.

One potential approach to treatment considers the use of small molecule drugs to
selectively suppress the normal proofreading function at PTCs, but not at normal
termination codons. This leads to a favorable competition of near-cognate
aminoacyl-tRNAs with the release factor and to the insertion of a near-cognate
amino acid at PTCs, allowing continued translation to full-length proteins. This
approach, also called “translational readthrough” or “suppression therapy”, was
first validated by using aminoglycoside (AG) antibiotics. Numerous in vitro and
in vivo experiments including clinical trials have demonstrated the ability of
selected structures of AGs (namely gentamicin, paromomycin and G418, Fig. 1) to
induce readthrough at PTCs and partially restore functional proteins. However,
severe side-effects of AGs, including high human toxicity, along with the
reduced readthrough efficiency at subtoxic doses, have limited their clinical
benefit for suppression therapy.

AGs selectively bind to the decoding A site on the 16S subunit of bacterial
rRNA, and kill bacteria by disturbing the fidelity of the decoding process.
Although prokaryotic selectivity is critical to their utility as antibiotics,
they are not perfectly selective for the bacterial ribosome; they also bind to
the eukaryotic A site resulting in PTC readthrough. Gentamicin and paromomycin
are three orders of magnitude more selective to the prokaryotic versus the
eukaryotic ribosome. For suppression therapy, this necessitates their use in
high quantity, which in turn causes deleterious toxic side-effects, and hence,
largely limits their utility.

A noteworthy exception is G418. In addition to its strong antibacterial
activity, it also exhibits the highest readthrough activity among all AGs tested
to date. G418 is however very cytotoxic to mammalian cells. It has not been
clear whether its high cytotoxicity is due to higher specificity to the
mammalian ribosome or to some other feature. Clearly, a systematic search for
new structures with improved PTC suppression activity and lower toxicity, along
with a deeper understanding on structure-activity-toxicity relationship, are
required to extrapolate the approach to the point where it can actually help
patients suffering from genetic diseases caused by nonsense mutations.

 

LOGO [g542149g0315200650055.jpg]

Towards these ends, we hypothesized that by separating the structural elements
of AGs that induce readthrough from those that affect toxicity we might obtain
potent AG-derivatives with improved readthrough activity and reduced toxicity.
By systematically fine-tuning the structure-activity-toxicity relationship, we
recently reported a series of structures, 1-8 (Fig. 2), exhibiting significantly
reduced toxicity and higher PTC suppression activity than either gentamicin or
paromomycin. Protein translation inhibition studies along with antibacterial
tests indicated that 1-8 have increased selectivity in their action towards
eukaryotic cells than towards prokaryotic cells in comparison to gentamicin and
paromomycin. However, none of those leads were able to outreach G418’s peak
suppression potency, nor its elevated eukaryotic specificity.

 

--------------------------------------------------------------------------------

Timor Baasov

 

The observed increased selectivity of action of 1-8 towards eukaryotic versus
prokaryotic ribosome along with their reduced toxicity drew our attention and
prompted us to ask several fundamental questions: what structural and
mechanistic features are responsible for the observed selectivity increase and
toxicity decrease of these synthetic derivatives? Can a general molecular
principle for their structure-activity-toxicity relationship be devised? Using
this principle, can a synthetic variant with similar or higher PTC suppression
activity and lowered toxicity than those of G418 be generated?

To address these questions, very recently we reported (see reference in J. Med.
Chem. 2012, in the publications list of the PI) on the design, synthesis and
evaluation of a new set of structures, 9-12 (Fig. 2) that perform better than
G418 by the above criteria while exhibiting lower toxicity. Furthermore, by
using a series of comparative readthrough, protein translation inhibition,
antibacterial and toxicity assays between standard and the entire set of
designer aminoglycosides 1-12, we demonstrated that the increased specificity
towards human cytoplasmic ribosome correlates with the increased PTC suppression
activity, and that the decreased specificity towards mitochondrial ribosome
confers, at least in part, to the lowered cell toxicity. These observations
provide proof of principle that antibacterial activity and toxicity of
aminoglycosides can be dissected from their suppression activity. The data
further indicated that AG-induced inhibition of cytoplasmic ribosome is a key
determinant for PTC suppression activity, and that the inhibition of
mitochondrial ribosome is key to AG-induced cell toxicity. These results are
therefore beneficial for further research on the development of AG-based drug
for the treatment of genetic diseases caused by nonsense mutations.

 

LOGO [g542149g0315200650180.jpg]

2. Market Survey

The National Institutes of Heanth (NIH) Office of Rare Diseases estimates that
genetic disorders are responsible for the majority of rare diseases and that
these diseases affect 25 million people in the US. Similar number of people is
also affected in European Union, (EU, estimated 29 million). Orphan diseases are
rare and often debilitating conditions, defined in the European Union (EU) as
having a prevalence of no more than five per 10,000 people. There are between
5,000 to 8,000 different rare diseases. It is estimated that, on average, 5-15%
of patients with any of at least 1,800 distinct genetic disorders have a
nonsense mutation as an underlying cause of the disease. Orphan drugs are those
medicines used in the diagnosis, prevention or treatment of orphan diseases.

Orphan drugs are a growing issue of importance to American and European
healthcare policy makers. The success of orphan drug legislation has resulted in
an increasing number of licensed medicines for rare diseases, and many more yet
unlicensed products have received orphan drug designation. Several studies
estimate a steady increase during 2010-2020 years in the cumulative number of
diseases for which an orphan drug is approved (Fig. 3), averaging just over 5
new diseases per year over the next 10 years. The annual per patient cost of
existing orphan drugs was seen to vary between €1,251 and €407,631, with the
median cost

 

5

 

--------------------------------------------------------------------------------

Timor Baasov

 

being €32,242 per year. The share of the total pharmaceutical market represented
by orphan drugs is predicted to increase from 3.3% in 2010 to a peak of 4.6% in
2016 after which it is expected to level off through 2020, as growth falls into
line with that in the wider pharmaceutical market.

Since to date CF is the most studied disease in this direction, we will focus on
this disorder in regard to the market of the potential drugs. Currently, more
than 1,000 different CF-causing mutations in the CFTR gene were identified, and
5-10% of the mutations are premature stop codons. In Ashkenazi Jews, the W1282X
mutation and other nonsense mutations account for 64% of all CFTR mutant
alleles. In the CF pipeline, there are promising new therapies designed to
rectify the cause of CF—a faulty gene and/or its faulty protein product. Bellow
is a “snapshot” of those potential CF therapies that are currently in
development.

GENE THERAPHY: Because a faulty gene causes CF, adding normal copies of the gene
to cells should correct these cells and ultimately cure the disease. Copernicus
Therapeutics, Inc. developed an approach that delivers normal copies of the CF
gene as tiny particles that slip into CF cells (the development is currently at
Phase 2).

PROTEIN ASSIST/REPAIR: This therapy is designed to correct the function of the
defective CFTR protein made by the CF gene to allow chloride and sodium to move
properly in cells lining in the lungs and other organs. This therapy is not
affected by the delivery challenges that have limited gene therapy and enzyme
replacement therapy. In addition, it does not necessitate the delivery of
foreign genetic material or viruses that gene therapy requires. It is
anticipated that by addressing the underlying cause of the disease, small
molecule drug might decrease dependence on palliative interventions and
ameliorate the debilitation and mortality in patients with genetic disorders. In
this category the following potential drugs are in pipeline:

a) PTC124 (PTC Therapeutics, Inc.) – The new drug candidate, PTC124, is designed
to repair one type of CF gene mutation, namely nonsense mutation that causes the
CFTR protein to stop being made in the cell before it is complete. PTC124 has
been on 2012 in phase 3 trial in CF and in DMD patients. These preliminary
results in patients with CF and DMD provide confirmation of proof-of-concept
that the compound that was originally designed by this company to suppress
premature stop mutations (PTC124) can indeed induce ribosomal readthrough of
nonsense mutations as an approach to treat genetic disorders.

 

LOGO [g542149g0315200650289.jpg]

b) VX-770 (Vertex Pharmaceuticals, Inc.) – This new compound is called a
“potentiator” and it may act upon the CFTR protein and help to open the chloride
channel in CF cells. Phase 3 dosing has been completed in patients, and since
November 2012 the compound is approved by FDA as a drug.

NBs vs PTC124: PTC124 also named as Ataluren (PTC Therapeutics, Inc. USA) is the
most advanced drug candidate investigated today for the treatment of genetic
defects resulting from nonsense mutations (Phase 3 in CF and DMD). However, yet
several problems are faced with the use of Ataluren for the suppression therapy:
(1) no real phenotype improvements have been seen after the clinical trials in
patients of both diseases; (2) the mechanism of its action is still not
conclusive; (3) its action as a readthrough inducer is somewhat controversial
since several labs reported its lack of action in different diseases models
while others reported opposite data.

In contrast, our developed structures 1-12 (Fig 2) belong to the same class of
aminoglycosides as gentamicin, but have no antibacterial activity, exhibit
significantly higher readthrough activity and lower toxicity than gentamicin and
they consistently demonstrated higher efficiency than that of gentamicin in a
series

 

6

 

--------------------------------------------------------------------------------

Timor Baasov

 

of diseases models both in vitro and in vivo tests: compound 2 in cellular and
animal models of CF (ref. no. 7 in LP), and cellular models of Rett syndrome
(refs. 5, 8); compounds 1 and 2 in cellular and in vivo models of USH1 (refs. 4,
11); compound 4 in cellular and animal models of HS (ref. 10). These
observations, together with the relatively low toxicity and high degree of
potency of the new generation structures 9-12 in targeting all six different
nonsense constructs underlying USH1, CF, DMD and HS, support the feasibility of
testing these novel AGs in treating these diseases in animal and human subjects.

3. Comprehensive Description of the Proposed Research

The data presented in the previous sections suggest that the newly developed
structures 1-12 (Fig. 2) also named NB-compounds (NBs) are worth for further
tests and development as drugs for treatment of cystic fibrosis and other
genetic diseases caused by nonsense mutations.

The main objectives of this research proposal are:

(1) to synthesize a large quantities of the selected NB-compounds, namely
compound 3 (NB74), compound 4 (NB84) and compound 9 (NB124), and supply them to
Eloxx company for the initial Eloxx evaluation to validate our published data on
this particular compounds;

(2) to further development NB-compounds for improved cellular uptake, increased
bioavailability and acute delivery.

 

LOGO [g542149g0315200650445.jpg]

Why we selected especially NB74, NB84 and NB124 for drug development? First,
previous studies have shown that the efficiency of aminoglycosides-induced
readthrough is highly dependent on: (i) the identity of stop codon (UGA > UAG >
UAA), (ii) the identity of the first nucleotide immediately downstream from the
stop codon (C > U > A ³ G) and (iii) the local sequence context around the stop
codon. Second, while in general the efficiency rank follows NB124 > NB84 > NB74,
it is not exactly true for all the mutations and the efficacy might be varied
significantly between different mutations for different compounds. For example,
our data on CF model shown in Fig. 4 (T. Baasov, S. Rowe and D. Bedwell,
unpublished data) clearly demonstrates that NB74 is more efficient than NB84 to
rescue the functional CFTR protein in primary bronchial epithelial cells of a CF
donor. Even though NB124 demonstrated highest activity, it is still somewhat
more toxic than NB84 and NB74 (Fig. 5), as demonstrated by comparative testing
of the potential ototoxoicity of these compounds on the mice cochlear explants
(T. Baasov and Jochen Schacht, unpublished data). It is clear that for the
patients with genetic

 

7

 

--------------------------------------------------------------------------------

Timor Baasov

 

diseases requiring a life-long treatment with these compounds, the potential
toxicity of these compounds is the most critical factor that limits their
further development as potential drug. Therefore, even though in general NB74 is
less active than NB84 and NB124, because its low toxicity is definitely worth
for further consideration for drug development.

Note that, because its low in vitro activity, NB74 has not pursued for long-term
in vivo animal studies. NB84 and NB124 have been evaluated in vivo in various
diseases models and have shown to be significantly better than gentamicin.

The proposed working plan includes:

Aim #1: According to our agreement my group will provide Eloxx the important
lead compounds NB74, NB84 and NB124, each compound in the quantities of up to
200 mg, chromatographically pure materials in the estimated time frame outlined
below. The stocks will undergo the desired biological tests including,
readthrough activity assay in dual luciferase reporter (standard USH-R3X
mutation) transfected in HEK cells and cytotixicity tests (HEK cells) to
validate the published data on these compounds on these specific assays.
Plasmids for readthrough activity assay and detailed synthesis and various
biological assay protocols will be supplied as well.

Aim #2: My group will continue further development of the lead compounds (NB74,
NB84 and NB124) to establish the formulation for increased bioavailability and
acute delivery (instead of systemic delivery, e.g. intravenous and/or
subcutaneous injection as they have been tested until to date). Shortly, because
aminoglycosides (AGs), like gentamicin and our leads NB82, NB74 and NB84 are
highly charged, water soluble compounds, they poorly absorb through intestinal
tissues and therefore are usually administered by injection. In addition, AGs
are short-lived molecules in the circulatory system, being rapidly eliminated by
glomerular filtration in the kidney. They also exhibit poor permeability into
eukaryotic cells, which requires their administration in higher dosages that in
turn causes harmful side effects and limits their use in translational therapy.
To solve these problems, we have initiated two different but complementary
directions.

Aim #2a: Chemical modification of NBs for increased lipophilicity and better
cellular uptake. To address this issue, we already initiated a project in which
we examined attachment of various alkyl/aryl groups on the pseudo-disaccharide
scaffold of lead structures (e.g. NB74 and NB124) at the N1 position and
generated four new scaffolds, compounds 13-16 (Fig. 6, T. Baasov, unpublished
data):

 

LOGO [g542149g0315200650617.jpg]

 

LOGO [g542149g0315200650539.jpg]

Interestingly, the readthrough activity of the previous pseudo-disaccharide
scaffolds, compounds 17 (NB82) and 18 (NB83) that provide the basic disaccharide
parts of NB74, NB124 (the compound 17) and of NB84 (the compound 18) have been
significantly improved. Especially noteworthy are the compounds 14 and 16,
exhibiting the propyl and benzyl group substitutions at N-1 position, which
exhibited highest activity. Compounds 14 and 16 were further tested for their
comparative activity against 17 and 18 in various diseases relevant reporter
systems (6 reporters that underline the diseases CF, DMD, USH and Hurler
syndrome), along with their inhibition of protein synthesis and toxicity (data
not shown) and found that these two scaffolds are indeed more active and less
toxic than the previously reported

 

8

 

--------------------------------------------------------------------------------

Timor Baasov

 

compounds 17 (NB82) and 18 (NB83). Based on these excellent preliminary data, we
aim to use these two newly developed scaffolds, compounds 14 (NB146) and 16
(NB145) to construct new generation of pseudo-trisaccharides similar to our
previous leads. These will be done by attachment of riboseamine and 5”-methyl
ribosamine at C5 position of the scaffolds to generate four new structures,
compounds 19-22 (Fig. 7). The synthesis of the new structures will be performed
by using a general tools and methodologies as described in our previous reports.
Once the new structures will be available they will be subjected to a series of
readthrough, translation inhibition and toxicity tests as these methodologies
are already well established in our laboratory.

Aim #2b: Development of new formulation of the lead structures NB74, NB84 and
NB124 for increased bioavailability and acute delivery. To address this issue we
have initiated a project in which we examined the formulation in which AGs are
microencapsulated into nano-cochleates through charge- charge interaction
between the AGs and lipid (phosphatidilserine-PS). We also developed an
efficient ELISA assay method to detect the encapsulated AG drug in cochleates
and/or in biological fluids (serum, urine, tissue, etc.). The preliminary data
on comparative readthrough activity of the clinical drugs gentamicin and G418
are attached below as Appendix 1. As can be seen from these data,
gentamicin-cochleate preparation is far better in terms of readthrough activity
than the corresponding gentamicn drug in solution. Further steps include: a) to
compare and contrast NB74, NB84 and NB124 in drug-cochleate versus solution in
terms of readthrough efficiency in dual luciferase assay protocol; b) the best
formulations from the previous step will be further evaluated for toxicity and
rescue of functional CFTR protein (in collaboration with the University of
Alabama at Birmingham) in cell lines and in CF-mouse models. c) To determine the
structure/size of the resulted drug-cochleate complexes by various spectroscopic
methods including cryo-electron-microscopy and freazfraction-microscopy
techniques. d) Continueous structure-activity relationship study to develop
appropriate size nano-cochleates with maximum activity and lowered toxicity
suitable for acute administration to treat genetic diseases (this part of the
project will be done in collaboration with Prof. Dganit Danino of the
Biotechnology and Food Engineering Department of Technion).

 

LOGO [g542149g0315200650726.jpg]

Note that the labs of Profs. Bedwell and Schacht are already tightly
collaborating with my lab and we have series of joint publications on this
subject (Bedwell: refs 7, 10 and Schacht: J.Med.Chem. 2009, 52, 2836- 2845).

4. Proposed Budget (in US Dollars) and Time Frame

 

    

Compounds

  

Time Frame

   Budget
Requested     

Remarks

  

Notes

Aim # 1                PI T. Baasov    ~200 mg NB74    2 weks-1 month    $ 8,000
     Rapid initial supply       ~200 mg NB84    2-3 months    $ 10,000     
Rapid initial supply       ~200 mg NB124    2-3 months    $ 12,000      Rapid
initial supply          Total Aim #1:    $ 30,000              

 

  

 

 

        Aim # 2a                   Synthesis of 19-22 and their evaluations   
8-12 months    $ 40,000         Aim # 2b                   Preliminary studies
for standard drug-cochleates SAR    4-6 months    $ 10,000      The cost of PS
and the synthesis of NBs are included       NB84-cochleates SAR    4-6 months   
$ 15,000               Total Aim #2:    $ 65,000              

 

  

 

 

             

Grand total for

the Aims # 1&2:

   $ 95,000              

 

  

 

 

       

 

9

 

--------------------------------------------------------------------------------

Timor Baasov

 

5. Detailed Budget (in US Dollars)

Personnel:

 

    

Role in project

  

    %

   Time      Salary  

1.

   Lab. Assistant    Lab. Assistant      50        12,000  

2.

   Postdoctorant    Researcher      50        12,000              

 

 

             Total:            24,000              

 

 

 

Supplies:

 

1. Chemicals, absolute & deuterated solvents

     28,000  

2. Lab. equipment, glassware, plastic ware

     12,000  

3. Biochemicals

     2,000  

4. In vitro and ex vivo assay kits

     10,000  

5. Chromatography material for various purifications

     13,000  

6. NMR and Mass Spectrometry tests

     6,000  

Total:

     71,000     

 

 

 

Grand Total:

   $  95,000     

 

 

 

Budget Justification:

The requests for laboratory assistant and postdoctorant for the duration of the
grant period are in recognition of the amount of work required in this project.
Considerable effort will be expended in the syntheses of various lead compounds
discussed in the proposal, their structure determination and analysis, assays
for their activity. The request for materials, supplies, and
chemicals/biochemicals is an important part for a successful development and
completion of the project.

6. Investigators’ Curriculum Vitae

Surname:                    Baasov                         First name:
            Timor

Birthdate:    January 3, 1954

(a) Education Background

 

From-To

  

Institution

  

Area of specialization

   Degree

1981-1986

   Weizmann Institute of Science    Chemistry    Ph. D.

1977-1979

   Tel-Aviv University    Chemistry    M. Sc.

1975-1977

   Tel-Aviv University    Chemistry    B. Sc.

Major research interest: Carbohydrate chemistry, Bioorganic and medicinal
chemistry, Drug design and development, Rational design of substrate and
inhibitors, Mechanistic enzymology.

 

10

 

--------------------------------------------------------------------------------

Timor Baasov

 

(b) Employment

 

From-To

  

Institution

  

Research area

   Title

2004-

   Technion    Bioorganic Chemistry    Professor

3/1998-8/1998

   The Scripps Research Institute, Cal    Bioorganic Chemistry    Visiting Prof.

1998-2003

   Technion    Bioorganic Chemistry    Assoc. Prof.

1990-1998

   Technion    Bioorganic Chemistry    Senior Lecturer

1988-1990

   Technion    Bioorganic Chemistry    Lecturer

1986-1988

   Harvard University    Bioorganic Chemistry    Post-Doct. Res.

 

11

 

--------------------------------------------------------------------------------

Timor Baasov

 

7. List of Publications (2010-2112)

 

1. I. Nudelman, D. Glikin, B. Smolkin, M. Hainrichson, V. Belakhov and T.
Baasov. Repairing faulty genes by amino glycosides: Development of new
derivatives of geneticin (G418) with enhanced suppression of diseases-causing
nonsense mutations. Bioorg. Med. Chem. 18, 3735-3746, (2010).

 

2. V. Pokrovskaya, I. Nudelman, J. Kandasamy and T. Baasov. Aminoglycosides:
Redesign Strategies for Improved Antibiotics and Compounds for Treatment of
Human Genetic Diseases. Methods in Enzymology. 478 (Glycomics), 437-462, (2010).

 

3. V. Pokrovskaya and T. Baasov. Dual-acting hybrid antibiotics: a promising
strategy to combat bacterial resistance. Expert Opinion in Drug Discovery. 5(9),
883-903, (2010).

 

4. T. Goldman, A. Rebibo-Sabbah, N. Overlack, I. Nudelman, V. Belakhov, T.
Baasov, T. Ben-Yosef, U. Wolfrum and K. Nagel-Wolfrum. Designed aminoglycoside
NB30 induces beneficial read-through of a USH1C nonsense mutation in the retina.
Investigative Ophthalmology & Visual Science, 51(12), 6671-6680, (2010).

 

5. C. Brendel, V. Belakhov, H. Werner, E. Wegener, J. Gaertner, I. Nudelman, T.
Baasov, P. Huppke. Readthrough of Nonsense Mutations in Rett Syndrome:
Evaluation of novel aminoglycosides and generation of a new mouse model. Journal
Molecular Medicine, 89, 389-398, (2011).

 

6. J. Kandasamy, D. Atia-Glikin, V. Belakhov, T. Baasov. Repairing faulty genes
by aminoglycosides: Identification of new pharmacophore with enhanced
suppression of diseases-causing nonsense mutations. Medicinal Chemistry
Communications, 2, 165-171 (2011).

 

7. S.M. Rowe, L.P. Tang, P. Sloane, K. Backer, M. Mazur, J. Buck;ey-Lauriel, I.
Nudelman, V. Belakhov, Z. Belok, E. Schwiebert, T. Baasov, D.M. Bedwell.
Suppression of CFTR Premature Termination Codons and Rescue of CFTR Protein and
Function by the Synthetic Aminoglycoside NB54. Journal Molecular Medicine
(Berl), 89, 1149-1154 (2011).

 

8. M. Vecsler, B. Ben Zeev, I. Nudelman, Y. Anikster, A. J. Simon, N. Amariglio,
G. Rechavi, T. Baasov, E. Gak. Ex Vivo Treatment with a Novel Synthetic
Aminoglycoside NB54 in Primary Fibroblasts from Rett Syndrome Patients
Suppresses MECP2 Nonsense Mutations. PLoS ONE, 6 (6), e20733 (2011).

 

9. H-L. R. Lee, C-C. Chen, T. Baasov, Y. Ron, J. P. Dougherty.
Post-transcriptionally Regulated Expression System in Human Xenogeneic
Transplantation Models. Molecular Therapy, 19(9), 1645-1655 (2011).

 

10. D. Wang , V. Belakhov, J. Kandasamy, T. Baasov, S-C. Li, Y-T Li, D.M.
Bedwell, K.M. Keeling. The designer aminoglycoside NB84 significantly reduces
glycosaminoglycan accumulation associated with MPS I-H in the Idua-W392X mouse.
Molecular Genetics and Metabolism 105, 116-125 (2012).

 

11. T. Goldmann, N. Overlack, F. Möller, V. Belakhov, M. van Wyk, T. Baasov, U.
Wolfrum, and K. Nagel-Wolfrum. A comparative evaluation of NB30, NB54 and PTC124
in translational read-through efficacy for treatment of an USH1C nonsense
mutation. EMBO Molecular Medicine, 4, 1-14, (2012).

 

12. J. Kandasamy, D. Atia-Glikin, E. Shulman, K. Shapira, M. Shavit, V. Belakhov
T. Baasov. Increased Selectivity toward Cytoplasmic versus Mitochondrial
Ribosome Confers Improved Efficiency of Synthetic Aminoglycosides in Fixing
Damaged Genes: A Strategy for Treatment of Genetic Diseases Caused by Nonsense
Mutations. J. Med. Chem. 55(23), 10630-10643 (2012).

 

13. M. Schalev, J. Kandasamy, N. Skalka, V. Belakhov, R. Rosin-Arbesfeld, T.
Baasov. Development of generic immunoassay for the detection of a series of
aminoglycosides with 6’-OH group for the treatment of genetic diseases in
biological samples. Journal of pharmaceutical and biomedical analysis. 75, 33-40
(2013).

 

14. K.M. Keeling, D. Wang, Y. Dai, S. Murugesan, B. Chenna, J. Clark; V.
Belakhov, J. Kandasamy, S.E. Velu, T. Baasov, D.M. Bedwell. Attenuation of
Nonsense-Mediated mRNA Decay Enhances In Vivo Nonsense Suppression. PLoS ONE 8
(4), e60478 (2013).

 

15. M. Schalev, J. Kondo, D. Kopelyanskiy, C.L. Jaffe, N. Adir, T. Baasov.
Identification of the molecular attributes required for Aminoglycoside activity
against Leishmania. PNAS 110 (33), 13333-13338 (2013).

 

16. X. Xue, V. Mutyam, L.P. Tang, S. Biswas, M. Du, L. A. Jackson, Y. Dai, V.
Belakhov, M. Shalev, F. Chen, J. Schacht, R. Bridges, T. Baasov, J. Hong, D. M.
Bedwell, S.M. Rowe. Synthetic Aminoglycosides Efficiently Suppress CFTR Nonsense
Mutations and Are Enhanced by Ivacaftor. Submitted (2013).

 

17.

 

12

 

--------------------------------------------------------------------------------

Appendix I

Comparative ex-vivo read through activity of aminoglycosides between the
solution and encapsulated within phosphatydylserine multilamelar structures
(cochleates)

 

LOGO [g542149g0315200650913.jpg]

Empty-C: Phosphatidyserine (PS) based cochleates has been prepared in the
absence of an aminoglycosidic agent (CaCl2 was used to replace the cation
required for cochleation procedure). Genta/G418: in solution. Genta-C/G-418-C:
Gentamicin or G418 were encapsulated within PS based cochleates.

Ex-vivo suppression of the R3X (USH1) mutation. The constructs of p2luc plasmid
harboring the R3X mutation were transfected to HEK-293 cells and addition of the
tested compounds was performed 6 h post transfection. Luciferase activity was
determined using the Dual Luciferase Reporter Assay System (Promega™).

Each bar represents the mean ± S.E.M. of 3 independent experiments (2 duplicated
each). Bars that are marked in an asterix (*) sign differ significantly at p <
0.05 according to a paired t test analysis.

 

--------------------------------------------------------------------------------

Timor Baasov

 

Appendix II

Synthetic schemes for the assembly of the developed leads NB74 and NB84 along
with of the required donor structure

Synthesis of NB74

 

LOGO [g542149g0315200651085.jpg]

 

14

 

--------------------------------------------------------------------------------

Timor Baasov

 

Synthesis of NB84

 

LOGO [g542149g0315200651272.jpg]

 

15

 

--------------------------------------------------------------------------------

Timor Baasov

 

Preparation of Trichloroacet-imidate DONOR (for both NB74 and NB84)

 

LOGO [g542149g0315200651397.jpg]

Note: All the synthetic procedures and analytical data for the above syntheses
of NB74, NB84 and NB124 are published in the Bioorganic and Medicinal Chemistry
Paper (2010) and JMC (2012).

 

16