Biomolecular Albumin-Oligonucleotide Assemblies

Modular Designs

We have introduced an albumin-ODN modular design [34] based on double stranded ODN annealing for site-specific incorporation of functional groups (Fig. 12.3). A panel of functionalised ODN modules can be synthesised and incorporated into the design by annealing to a complementary ODN "handle” strand positioned at Cys34 on the albumin molecule (Fig. 12.3A). Attachment of functional groups to the 3' and 5' termini of the complementary ODN (cODN) and the free termini of the ODN "handle” offers the possibility for combinatorial attachment of three functional groups with a simple double stranded DNA duplex design (Fig. 12.3B). This number can be potentially expanded by incorporation of functionalised nucleotides into the ODN sequence. Furthermore, the inter-molecule distance can be controlled by altering the length of the ODN sequence and the position of inserted nucleotides. The approach allows incorporation of the functional molecules onto ODN precursor strands before attachment to albumin or the annealing process. This offers the potential for "off-the-shelf" selection of cODN modules bearing a functionality specific to the therapeutic or diagnostic requirement.

Albumin-Oligonucleotide Constructs

For our approach, an initial requirement is the preparation of a recombinant human albumin/ODN construct (rHAODN) by a two- step procedure. Step 1: maleimide functionalisation ("activation") of a 21-mer amine-modified ODN with a bifunctional succinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate (SMCC) linker. Step 2: SMCC-activated ODN fractions, purified by reverse-phase high-performance liquid-chromatography, are then reacted to form the rHA/ODN construct that is verified by MALDI-ToF analysis. The conjugation efficiency correlated with the number of free Cys34 thiols on albumin quantified by an Ellman's assay, and was not influenced whether a 3' or 5'-SMCC-activated ODN is reacted, or a molecule is attached at its opposite termini. This offers the possibility to use the available free 3' or 5' end on the ODN "handle” for incorporation of an extra functional group in addition to those added following annealing with a cODN. The assembly with a fluorescent Atto488-bearing ODN complementary sequence into a rHA/ODN construct was demonstrated with gel electrophoresis, with base-pair complementarity required for co-localisation of the Atto488 fluorescent signal and Coomassie-stained albumin (Fig. 12.4).

Schematic representation of biomolecular albumin-oligonucleotide assemblies exemplified with a double stranded oligodeoxynucleotide

Figure 12.3 Schematic representation of biomolecular albumin-oligonucleotide assemblies exemplified with a double stranded oligodeoxynucleotide (ODN) design. A. Single stranded ODN "handle" attached by covalent interaction to the Cys34 position of recombinant human albumin is a substrate for reaction with a functional group attached to the termini of complementary ODN (clockwise; aptamer, nanobody, radionucleotide and fluorophore). Base- pair driven annealing results in modular self-assembly and site-specific functionalisation of the albumin. B. Termini functionality at both the 3' and 5' end allows precise attachment of three functional groups. Graphic prepared by Simon Lykkemark, Aarhus University.

371

Biomolecular Albumin-Oligonucleotide Assemblies

SOS gel electrophoresis analysis of rHAODN3 and cODNS* annealing

Figure 12.4 SOS gel electrophoresis analysis of rHAODN3 and cODNS* annealing. The gel was stained for ODN/cODN detection with SYBR gold (nucleotide) and for protein detection with Coomassie staining (protein). The labelled complementary strand cODN5* was detected by Atto488 fluorescence. The fluorophore detection and protein detection pictures were merged (merge). rHAODN6 and cODN5* annealing experiments were used as non-specific annealing negative control (nc) as ODN6 and cODN5* bear the same sequence. Annealing was performed with rHAODN3:cODN5* ratios of 1:0.5, 1:1, and 1:2. Free rHA was not separated from the rHAODN material before the analysis. The experiment represents one of three independent similar experiments. Figure reproduced with permission from Mol Ther Nucleic Acids. 2017; 9: 284-293.

Application for Half-Life Extension of Nucleic Acid Aptamers

Nucleic acid aptamers are single stranded DNA or RNA designed to adopt secondary and tertiary structures through base pair-driven folding that can interact with high specificity to cognate targets [35,

36]. This has promoted them as targeting agents or molecular drugs [37-39]. The rapid renal clearance due to the small size ~ 5-15 kDa, however, requires enabling technologies to increase residence time in the bloodstream. Attachment of the synthetic polymer poly (ethylene glycol) (PEG) is a conventional drug half-life extension approach, however, immune responses to PEG have been reported [40, 41]. PEGylated half-life extension technology has been used to increase the blood circulation of a factor IXa (FIXa) blocking anticoagulant nucleic acid aptamer [42, 43]; however, allergic reactions were observed in clinical trials attributed to possible immune responses to the PEG polymer [44].

The aforementioned long circulatory property utilised in a number of marketed products, promotes albumin as an attractive alternative to PEG, that we have used as an enabling technology for a FIXa aptamer [34].

Thealbumin-oligonucleotide-aptamer (rHAODN//apt) assembly process was facilitated using a FIXa aptamer design (cODNaptamer) that contains an extended DNA sequence complementary to the ODN "handle” coupled to albumin (rHAODN). The aptamer was positioned distal to the rHA site to minimise any possible steric hindrance to aptamer target engagement. 2'-fluoro and 2'-0-methyl RNA modifications were incorporated into the aptamer design to mediate stability to serum nucleases. rHAODN//apt samples were purified from non-incorporated cODNaptamer and rHAODN by size- exclusion chromatography. A requirement for the albumin-based aptamer construct design is retained albumin (FcRn) and aptamer (FIXa) target engagement to allow concomitant cellular recycling and anti-coagulant activities, respectively. Exploitation of the coagulation cascade for factor IXa-mediated conversion of factor X (FX) to factor Xa (FXa) was used as an assay to measure FXIa activity after addition of rHAODN//anti-FIXa apt. Addition of increased concentrations of rHAODN//apt to FIXa resulted in a dose-dependent reduction of FXa determined by reduced levels of a FXa substrate cleavage product (Fig. 12.5). This suggests engagement and blocking of FIXa by the anti-FIXa aptamer is maintained following assembly into the rHAODN//apt biomolecular construct.

373

Biomolecular Albumin-Oligonucleotide Assemblies

FIXa activity assay with rHAODN//apt

Figure 12.5 FIXa activity assay with rHAODN//apt. Substrate development (RFU) by FXa was monitored over time after incubation with FIXa. Preincubation of FIXa with the rHAODN//apt construct resulted in a lower activity of FXa (a reduced slope) indicating dose-dependent inhibitory activity towards FlXa- mediated conversion of FX to FXa. The figure shows the average and standard deviations of three experiments. Figure reproduced with permission from Mol Ther Nucleic Acids. 2017; 9: 284-29S).

We have previously observed a reduction in FcRn engagement following molecule attachment to wild type albumin, even if positioned at the Cys34 site in DI distant from the predominant albumin/FcRn Dill binding interface [45, 46]. This is most likely due to molecule-induced steric hindrance in a contributory FcRn binding interface lying in DI shown to be required for optimal FcRn binding [47]. In accordance with our previous findings with alternative molecules, attachment of the ODN assembly at Cys34 similarly resulted in a decrease (~9x after ODN conjugation) of FcRn binding compared to non-modified wild type albumin (Table 12.1). In our previous work [45, 46] conjugation to a recombinant human albumin variant engineered for high FcRn binding was used to rescue the FcRn-binding capacity. The high FcRn- binding variant (HB) exhibited almost lOx higher FcRn binding than the wild type albumin (Table 12.1). This was successfully translated to the biomolecular constructs with the rHAODN//apt assembly containing the HB exhibiting approximately 30x and 3x higher FcRn binding affinity (Dissociation constant (ffD) 0.28 ± 0.08 pM) compared to the wild type rHAODN//apt construct 8.80 ± 2.17 pM) and non-modified wild type albumin control (ffD 0.98 ± 0.25 pM), respectively. Inclusion of albumin variants engineered for high FcRn variants, thus, can be potentially used to increase the circulatory half-life of albumin-nucleic acid biomolecular assemblies.

Table 12.1 Biolayer Interferometry hFcRn Affinity Binding Studies of rHA, Annealed rHAODN//apt, hFcRn HB, and Annealed HBODN//apt

KD (f>M)

kon x 103 (1 Ms’1)

k0ff x 10"3 (1 s’1)

rHA

0.98 ±0.25

11.2 ± 1.96

10.6 ±0.64

rHAODN//

apt

8.80 ±2.17

1.73 ±0.31

14.8 ± 1.00

HB

0.10 ±0.008

10.8 ±0.14

1.04 ±0.10

HBODN//apt

0.28 ±0.08

3.32 ±0.31

0.95 ±0.32

Note. The KD-, k0„- and koff-values are averages of 3 measurements, each obtained using a 5-step dilution series from 0.1875-3.0 pM at pH 5.5 and for rHAODN//apt a 6 step dilution from 0.1875 to 6.0 pM. All curves are fitted to a 1:1 binding model with the relation KD = к^/к0„.

Table reproduced with permission from Mol Ther Nucleic Acids 2017; 9: 284-293.

 
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