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Table 2 Summary of the efficacy of the different intrabodies against HD

From: The P42 peptide and Peptide-based therapies for Huntington’s disease

Antibody Target of the peptide Model Population Way of administration End point Method of evaluation Results
C4 intrabody N17 terminal region Cell culture (Lecerf et al., 2001) [44] COS-7; BHK-21; HEK293 Co-transfection: hHtt17aa-25/73/103Q-GFP ± C4 intrabody (ratio 5:1) Aggregation % of aggregate-positive transfected cells Reduction up to 86 %
Organotypic cultures (Murphy and Messer, 2004) [46] Cortico-striatal slice cultures Malonate treatment, and transfection with hHtt17aa-25/72Q-GFP ± C4 intrabody Cell survival % of co-transfected died or dying cells Rescue to wt level
Drosophila model (Wolfgang et al., 2005) [45] ELAV-Gal4; UAS-hHttex1-20/93Q; Genetic cross: UAS-C4 intrabody” Survival % of survival to adult (eclosion); mean, median, and maximal lifespan Increased survival to adulthood (from 23 % to 100 %); increased mean adult lifespan by 30-50 %
Aggregation; quantification of soluble polyQ forms Immunostaining; detergent-soluble hHttex1-93Q detection (Western blot) Slowing of visible aggregate formation. Increased levels of soluble Htt
Neurodegeneration Photoreceptors/ommatidium quantification Slowing of neurodegeneration in photoreceptors cells
Mouse model (Snyder-Keller et al., 2010) [47] B6.HD6/1 125Q (hHttex1-125Q) C4 intrabody with AAV vector into the striatum; presymptomatic (injection: wk5 to 8 ; killed at wk16 to 32); symptomatic (injection wk 10 to 24; killed 8 to 10 wk later) Aggregation Immunostainig: number and size aggregates Pre-symptomatic and symptomatic effect: aggregate reduction (size > number), more important in pre-symptomatically treated mice
VL12.3 intrabody N17 terminal region Cell culture (Colby et al., 2004) [48] HEK293 Co-tranfection: hHttex1-97Q-GFP + empty vector or VL12.3 Aggregation Immunostaining 50 % reduction of aggregates vs empty vector
Cell culture (Southwell et al., 2008) [49] HEK293 Co-transfection: hHttex1-103Q-GFP + empty vector or VL12.3 Aggregation Immunostaining Dose-dependent aggregate reduction
Cell survival % of co-transfected dead cells Reduced cell toxicity
Co-transfection: hHttex1-25/103Q-GFP + VL12.3 Quantification of soluble and insoluble hHttex1 Centrifugation and Immunoblot assay Significant reduction of insoluble but not of soluble hHttex1-103Q levels
Co-transfection: hHttex1-103Q -SNAP tag ± VL12.3 hHttex1-103Q turnover Fluorescence intensity of SNAP-tag No effect on polyQ turnover
Cortico-striatal brain slice model (Southwell et al., 2008) [49] Rat brain slices Co-transfection: YFP as morphometric marker ± hHttex1-103Q -CFP ± VL12.3 Neurodegeneration Immunostaining: counting of healthy striatal medium spiny neurons (MSNs) Rescue of neurodegeneration at the level of wt cells
ST14A striatal precursor cells Co-transfection: hHttex1-103Q -GFP ± VL12.3 hHttex1-103Q localisation and turnover Immunostaining: cytoplasmic/nuclear hHttex1-103Q ratio Altered cytoplasmic/nuclear trafficking: significant increase of nuclear Htt
Mouse model (Southwell et al., 2009) [51] C57BL/6 (lentiviral HD model) HD model: Unilateral striatal injection: hHttex1-103Q -GFP or GFP lentivirus.Treatment: + VL12.3- AAV or GFP (4 wk-old mice). Tests 6wks later. Amphetamine-induced rotation Ipsilateral rotations tested during 30’ after intraperitoneal amphetamine injection. Strong reduction of the number of ipsilateral rotations to the levels of GFP lentivirus injected animals
MSNs loss DARPP-32 staining Rescue to the levels of GFP lentivirus injected animals
Aggregation Striatal immunostaining with anti-Htt MW8 (detect aggregates only) Significant aggregate reduction vs AAV-GFP injected animals
YAC128 (Full length-hHtt-128Q) 2-months-old male mice and wt littermates injected bilaterally in the striatum with GFP- or VL12.3- AAV Motor performances Rotarod latency to fall (monthly from 3 to 7 months of age) No effect
Beam-crossing performance (monthly from 3 to 7 months of age) No effect
Climbing time (7-month-old mice) No effect
Cognitive performances (spatial and cortical learning) Novel object location and novel object preference tests (7-month-old mice) No effect in both tests
Anxiety Open field test Non significant amelioration
Brain atrophy Ventricular size assessment (7-month-old mice) No effect
Body weight Assessment monthly from 3 to 7 months of age No effect
R6/2 (hhttex1- 144Q) 3-day-old male mice and wt littermates: bilateral injection at the center of each forebrain hemisphere of GFP- or VL12.3-AAV Motor performances Rotarod latency to fall (weekly from w4 to death) Reduced latency to fall (wk 10 to 12)
Beam-crossing performance (weekly from w4 to death) No rescue: Increased severity of the phenotype (time to cross the beam)
Brain atrophy Ventricular size assessment (10-wk-old mice) No effect
Body weight Assessment weekly from 4 wk until death No effect
Aggregation Striatal immunostaining with anti-Htt MW8 (detect aggregates only) and nuclear marker; counting of positive foci (10 week-old mice) Reduction of the number of neuropil aggregates; no significant reduction of intranuclear aggregates
Life span Once ill, twice a day assessment of righting reflex Aggravation and decrease survival
MW7 intrabody Poly P region Cell culture (Khoshnan et al., 2002) [50] HEK293 Co-transfection: hHttex1-97Q-GFP and MW7 or empty vector Aggregated/soluble Htt Centrifugation, SDS treatment and western blotting Reduction of both aggregated and soluble polyQHtt
Cell survival TUNEL staining 33 % reduction of TUNEL positive cells
Cell culture (Southwell et al., 2008) [49] HEK293 Co-transfection: hHttex1-103Q-GFP + empty vector or MW7 Aggregation Immunostaining Aggregate reduction with a threshold-effect
Cell survival % of co-transfected dead cells Reduced cell toxicity
Co-transfection: hHttex1-25/103Q-GFP + MW7 Quantification of soluble and insoluble hHttex1 Centrifugation and Immunoblot essay Significant reduction of both soluble and insoluble hHttex1-103Q; no effect on soluble wt hHttex1-25Q
Co-transfection: hHttex1-103Q-SNAP tag ± MW7 hHttex1-103Q turnover Fluorescence intensity of SNAP tag Significant decreased fluorescence (increased hHttex1-103Q turnover)
Cortico-striatal brain slice model (Southwell et al., 2008) [49] Rat brain slices Co-transfection: YFP ± hHttex1-103Q -CFP ± MW7 Neurodegeneration Immunostaining: counting of healthy MSNs Non-significant reduction of neurodegeneration
ST14A striatal precursor Co-transfection: hHttex1-103Q -GFP ± MW7 hHttex1-103Q localisation and turnover Immunostaining: cytoplasmic/nuclear hHttex1-103Q ratio No effect
Happ1-Happ3 antibodies Poly P region Cell culture (Southwell 2008) [49] HEK293 Co-transfection: hHttex1-103Q-GFP + empty vector or Happ1-Happ3 Aggregation Immunostaining Dose-dependent aggregate reduction
Cell survival % of co-transfected dead cells Reduced cell toxicity
Co-transfection: hHttex1-25/103Q-GFP + Happ1-Happ3 Quantification of soluble and insoluble hHttex1 Centrifugation and Immunoblot essay Significant reduction of both soluble and insoluble hHttex1-103Q; no effect on soluble wt hHttex1-25Q
Co-transfection: hHttex1-103Q-SNAP tag ± Happ1-Happ3 hHttex1-103Q turnover Fluorescence intensity of SNAP tag Significant decreased fluorescence (increased hHttex1-103Q turnover)
Cortico-striatal brain slice model (Southwell et al., 2008) [49] Rat brain slices Co-transfection: YFP as morphometric marker ± hHttex1-103Q -CFP ± Happ1-Happ3 Neurodegeneration Immunostaining: counting of MSNs Significant reduction of neurodegeneration
ST14A striatal precursor Co-transfection: hHttex1-103Q -GFP ± Happ1-Happ3 hHttex1-103Q localisation and turnover Immunostaining: cytoplasmic/nuclear hHttex1-103Q ratio No effect
Mouse model (Southwell et al., 2009) [51] C57BL/6 (lentiviral HD model) HD model: Unilateral striatal injection: hHttex1-103Q -GFP or GFP lentivirus.Treatment: + GFP- or Happ1- AAV (4 wk-old mice). Tests 6wks later. Amphetamine-induced rotation, Ipsilateral rotations tested during 30’ after intraperitoneal amphetamine injection. Strong reduction of the number of ipsilateral rotations to the levels of GFP lentivirus injected animals
MSNs loss DARPP-32 staining Rescue to the levels of GFP lentivirus injected animals
Aggregation Striatal immunostaining with anti-Htt MW8 (detect aggregates only) and nuclear marker; counting of positive foci. Significant aggregate reduction vs AAV-GFP injected animals
YAC128 (Full length-hHtt-128Q) 2-months-old male mice and wt littermates injected bilaterally in the striatum with GFP- or Happ1- AAV Motor performances Rotarod latency to fall (monthly from 3 to 7 months of age) Improvement in 3-, 4-, and 7 –month-old mice
Beam-crossing performance (monthly from 3 to 7 months of age) Partial improvement
Climbing (7-month-old mice) Increased climbing time to the level of wt littermates
Cognitive performances (spatial and cortical learning) Novel object location and novel object preference tests (7-month-old mice) Significant amelioration of spatial and cortical learning
Anxiety Open field test Rescue to the level of wt littermates
Brain atrophy Ventricular size assessment (7-month-old mice) Reduction of ventricular size
Body weight Assessment monthly from 3 to 7 months of age No effect
R6/2 (hhttex1- 144Q) 3-day-old male mice and wt littermates: bilateral injection at the center of each forebrain hemisphere of GFP- or Happ-AAV Motor performances Rotarod latency to fall (weekly from 4 wk until death) Amelioration (between w9 and 12 of age) vs GFP-AVV injected animals.
Beam-crossing performance (weekly from 4 wk until death) Reduction of the time to cross the 12 mm beam in 10- and 11-week-old mice, and the 6 mm beam between 9 and 11 weeks of age
Brain atrophy Ventricular size assessment (10-wk-old mice) Reduction of ventricular size to the level of wt littermates
Body weight Assessment weekly from 4 wk until death No effect
Aggregation Striatal immunostaining with anti-Htt MW8 (detect aggregates only) and nuclear marker; counting of positive foci (10 week-old mice). Reduction of the number of both neuropil and intranuclear aggregates.
Life span Once ill, twice a day assessment of righting reflex No effect
N171-82Q: hHtt171aa-82Q Four-week old male mice and wt littermates: bilateral striatal injection of GFP- or Happ1-AAV Motor performances Latency to fall with accelerating rotarod (every 2 wks from wk6 until death) Significant improvement from wk 20 to 40 at the level of wt mice
Beam-crossing performance (every 2 wks from wk6 until death) Significant improvement with reduction of time to cross the three beams vs GFP-AAV treated and wt mice.
Clasping (22-week-old mice) Attenuation of clasping behavior
Body weight Assessment every 2 wks from wk6 until death Increased weight vs GFP-AAV treated but not to the level of wt littermates (from wk 22)
Life span Once ill, twice a day assessment of righting reflex 33 % increase of maximum life-span (from 30 to 40 wk) vs GFP-AAV treated mice.
BACHD: Full-length- hHtt-97Q- 2-months-old male mice and wt littermates: bilateral striatal injection of GFP- or Happ1-AAV Motor performances Rotarod latency to fall (monthly from month 3 to 6) Increased latency to fall in 5- and 6- month-old mice
Beam-crossing performances (monthly from month 3 to 6) Decrease time to cross the beams at 5 and 6 months (28 mm beam) and at month 6 (6 mm beam)
Climbing time (6-month-old mice) Increased climbing time vs GFP treatment
Cognitive performances (spatial and cortical learning) Novel object location and novel object preference tests (6-month-old mice) No effect
Anxiety Open field test Significant effect vs GFP-AAV treated mice
Brain atrophy Ventricular size assessment (6-month-old mice) Reduction of ventricular size
Body weight Assessment monthly (from 3 to 6 months of age) No effect
mEM48 intrabody (Wang et al., 2008) [52] VA residues after the polyP region Cell culture HEK293 Co-transfection: hHtt208aa 23/130Q ± EM48 Cell survival % of co-transfected dead cells Improved cell viability
Rat cortical neurons Co-transfection: hHtt208aa 23/130Q ± EM48 Neuritic disruption and pyknotic nuclei Neuronal morphology Significant reduction of transfected neurons with disrupted neurites or fragmented nuclei
PC12 cells Transfection of hHtt208aa 23/130Q ± AAV-EM48 Neuropil aggregates Immunostaining Significant reduction of neuropil aggregates
Mouse model R6/2 (hhttex1- 144Q) Intrastriatal injection of helper dependent AAV EM48 (7-wk-old mice) Neuropil aggregates Immunostaining (4 wk after injection) Significant less neuropil aggregates vs non injected region; no effect on intranuclear inclusion
N171-82Q Bilateral striatal injection of helper dependent AAV EM48 (10-wk-old mice) Neuropil aggregates Immunostaining (6 wk after injection) Significant less neuropil aggregates vs non injected region; no effect on intranuclear inclusions
Motor performances Stride length (8-wk post injection) Improvement
Rotarod latency to fall (8-wk post injection) Significant improvement
Body weight   No effect
Survival   No effect
Monoclonal antibodies 1C2 (Heiser et al., 2000 [43]; Trottier et al., 1995) [42] PolyQ chain (soluble) Cell culture COS-1 Co-transfection: hHttex1-51Q ± 1C2 Aggregation Filter retardation assay Up to 85 % reduction in aggregates
  1. Legend: To characterize Htt fragments we use the general indication HttXaa-YQ: the length of the fragment is expressed as a number X of amino acids (aa) (superimposed); the length of polyQ expansion is expressed as a number Y of Q.